Crystalline polymorphs of the free base of 2-hydroxy-6-((2-(1-isopropyl-1h-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde

ABSTRACT

Disclosed are crystalline free base ansolvate forms of 2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde (or Compound 1), such as the free base Form I, Form II and Material N. Also disclosed are crystalline free base solvates of 2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde (or Compound 1).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/937,393 filed Feb. 7, 2014, and U.S. Provisional Application No.61/937,404 filed Feb. 7, 2014, the contents of each of which isincorporated herein in its entirety by reference

BACKGROUND

2-Hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehydeis a compound having the formula:

Sickle cell disease is a disorder of the red blood cells, foundparticularly among those of African and Mediterranean descent. The basisfor sickle cell disease is found in sickle hemoglobin (HbS), whichcontains a point mutation relative to the prevalent peptide sequence ofhemoglobin (Hb).

Hemoglobin (Hb) transports oxygen molecules from the lungs to varioustissues and organs throughout the body. Hemoglobin binds and releasesoxygen through conformational changes. Sickle hemoglobin (HbS) containsa point mutation where glutamic acid is replaced with valine, allowingHbS to become susceptible to polymerization to give the HbS containingred blood cells their characteristic sickle shape. The sickled cells arealso more rigid than normal red blood cells, and their lack offlexibility can lead to blockage of blood vessels. A need exists fortherapeutics that can treat disorders that are mediated by Hb or byabnormal Hb such as HbS, such as2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde.

When used for treating humans, it is important that a crystalline formof a therapeutic agent, like2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde,or a salt thereof, retains its polymorphic and chemical stability,solubility, and other physicochemical properties over time and amongvarious manufactured batches of the agent. If the physicochemicalproperties vary with time and among batches, the administration of atherapeutically effective dose becomes problematic and may lead to toxicside effects or to ineffective therapy, particularly if a givenpolymorph decomposes prior to use, to a less active, inactive, or toxiccompound. Therefore, it is important to choose a form of the crystallineagent that is stable, is manufactured reproducibly, and hasphysicochemical properties favorable for its use as a therapeutic agent.

However, the art remains unable to predict which crystalline form of anagent will have a combination of the desired properties and will besuitable for human administration, and how to make the agent in such acrystalline form.

SUMMARY Ansolvates

This invention arises in part out the discovery that an HCl salt ofCompound 1 disproportionates or loses HCl, and a disproportionation ofthe HCl salt of Compound 1 in water generates the free base anddisproportionation was facile upon exposure to elevated humidity, withwet milling, and in direct contact with water (e.g. slurry). The sulfatesalt of Compound 1 also disproportionates from certain solvents such asdimethyl sulfoxide and methanol when precipitated with water. Thevolatilization of HCl was evident within hours of exposure to dryingconditions. For example, partial conversion to the free base wasobserved within 12 hours at 30° C. Accordingly, the free base ofCompound 1 provides a stabler chemical entity compared to thecorresponding HCl or sulfate and such other salt.

It has now been discovered that2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde(or Compound 1) i.e., the free base of Compound 1, can be obtained asone or more crystalline ansolvate forms, several of which are referredto here as crystalline Form I, Form II and Material N. In preferredembodiments, the free base of Compound 1 is a crystalline ansolvate,such as a crystalline anhydrous form. The free base of Compound 1, canbe obtained from its corresponding salt form, such as the HCl salt ofCompound 1.

Three anhydrous crystalline forms of the free base were identified,termed Free Base Forms I, II, and Material N. It has been discoveredthat nucleation of Free Base Form I generally occurs first from aslurry. Extending the slurry time can induce the transformation of FreeBase Form I to Free Base Form II, a thermodynamically more stable phaserelative to Form I. It has further been discovered that Free BaseMaterial N can be stable relative to Forms I and II, at roomtemperature.

Free Base Material N was found to be enantiotropically related to FormII, and will transform reversibly at a specific transition temperature(estimated herein near 40-42° C.). Above the transition temperature,Free Base Form II appears to be the most stable form, relative to Form Iand Material N. Thus, under operating temperatures below 40° C., e.g.,at 30° C., the free base of Compound 1 exists primarily as Material N,which may have some residual Form II. Thus, at operating temperaturesabove 40° C., e.g., at 50° C., the free base of Compound 1 existsprimarily as Form II, which may have some residual Material N. At 40° C.little appreciable conversion is seen between Material N and Form II.This is contemplated to be true for slurries of the free base in certainsolvents and in the solid state. In one embodiment, the one or morecrystalline free base forms of Compound 1 do not undergo polymorphictransformation under conditions suitable for manufacturing and storingthe crystalline forms.

Form I

In one embodiment, the crystalline free base of Compound 1 comprisescrystalline Form I, which is characterized by an endothermic peak at(97±2) C as measured by differential scanning calorimetry. In anotherembodiment, the crystalline Form I of the free base of crystallineCompound 1 is characterized by the substantial absence of thermal eventsat temperatures below the endothermic peak at (97±2) ° C. as measured bydifferential scanning calorimetry. In another embodiment, thecrystalline Form I of the free base of crystalline Compound 1 ischaracterized by an X-ray powder diffraction peak (Cu Kα radiation atone or more of 12.82°, 15.74°, 16.03°, 16.63°, 17.60°, 25.14°, 25.82°and 26.44°±0.2 °2θ. In another embodiment, the crystalline Form I of thefree base of crystalline Compound 1 is characterized by an X-ray powderdiffraction pattern (Cu Kα radiation) substantially similar to that ofFIG. 3.

In another embodiment, the crystalline Form I of the free base ofcrystalline Compound 1 is characterized by at least one X-ray powderdiffraction peak (Cu Kα radiation) selected from 12.82°, 15.74°, 16.03°,16.63°, 17.60°, 25.14°, 25.82° and 26.44° (each ±0.2 °2θ). In anotherembodiment, the crystalline Form I of the free base of crystallineCompound 1 is characterized by at least two X-ray powder diffractionpeaks (Cu Kα radiation) selected from 12.82°, 15.74°, 16.03°, 16.63°,17.60°, 25.14°, 25.82° and 26.44° (each ±0.2 °2θ). In anotherembodiment, the crystalline Form I of the free base of crystallineCompound 1 is characterized by at least three X-ray powder diffractionpeaks (Cu Kα radiation) selected from 12.82°, 15.74°, 16.03°, 16.63°,17.60°, 25.14°, 25.82° and 26.44° (each ±0.2 °2θ).

In another embodiment, Form I is characterized by 1, 2, 3, 4, or morepeaks as tabulated below.

Observed Peaks for Form I, XRPD File 609973.

°2θ d space (Å) Intensity (%)  5.52 ± 0.20 16.021 ± 0.602  68 12.82 ±0.20 6.906 ± 0.109 74 15.03 ± 0.20 5.897 ± 0.079 38 15.74 ± 0.20 5.629 ±0.072 46 16.03 ± 0.20 5.530 ± 0.069 46 16.63 ± 0.20 5.331 ± 0.064 6117.60 ± 0.20 5.040 ± 0.057 100 18.74 ± 0.20 4.736 ± 0.051 24 19.07 ±0.20 4.654 ± 0.049 17 19.35 ± 0.20 4.587 ± 0.047 23 20.32 ± 0.20 4.370 ±0.043 18 21.64 ± 0.20 4.106 ± 0.038 23 22.80 ± 0.20 3.901 ± 0.034 2623.28 ± 0.20 3.821 ± 0.033 34 25.14 ± 0.20 3.543 ± 0.028 52 25.82 ± 0.203.451 ± 0.026 81 26.44 ± 0.20 3.371 ± 0.025 51 27.91 ± 0.20 3.197 ±0.023 17 28.19 ± 0.20 3.165 ± 0.022 26

Form II

In another embodiment, the crystalline Compound 1 free base comprisescrystalline Form II, which is characterized by an endothermic peak at(97±2) C as measured by differential scanning calorimetry. In anotherembodiment, the crystalline Form II of the free base of crystallineCompound 1 is characterized by the substantial absence of thermal eventsat temperatures below the endothermic peak at (97±2) ° C. as measured bydifferential scanning calorimetry. In another embodiment, thecrystalline Form II of the free base of crystalline Compound 1 ischaracterized by an X-ray powder diffraction peak (Cu Kα radiation atone or more of 13.37°, 14.37°, 19.95° or 23.92 °2θ. In anotherembodiment, the crystalline Form II of the free base of crystallineCompound 1 is characterized by an X-ray powder diffraction pattern (CuKα radiation) substantially similar to that of FIG. 5.

In another embodiment, the crystalline Form II of the free base ofcrystalline Compound 1 is characterized by at least one X-ray powderdiffraction peak (Cu Kα radiation) selected from 13.37°, 14.37°, 19.95°and 23.92° 02 (each ±0.2 °2θ). In another embodiment, the crystallineForm II of the free base of crystalline Compound 1 is characterized byat least two X-ray powder diffraction peaks (Cu Kα radiation) selectedfrom 13.37°, 14.37°, 19.95° and 23.92° 02 (each ±0.2 °2θ). In anotherembodiment, the crystalline Form II of the free base of crystallineCompound 1 is characterized by at least three X-ray powder diffractionpeaks (Cu Kα radiation) selected from 13.37°, 14.37°, 19.95° and 23.92°02 (each ±0.2 °2θ).

In another embodiment, Form II is characterized by 1, 2, 3, 4, or morepeaks as tabulated below.

Observed Peaks for Form II, XRPD File 613881.

°2θ d space (Å) Intensity (%)  5.62 ± 0.20 15.735 ± 0.581  24 12.85 ±0.20 6.888 ± 0.108 22 12.97 ± 0.20 6.826 ± 0.106 21 13.37 ± 0.20 6.622 ±0.100 100 14.37 ± 0.20 6.162 ± 0.087 56 15.31 ± 0.20 5.788 ± 0.076 2116.09 ± 0.20 5.507 ± 0.069 23 16.45 ± 0.20 5.390 ± 0.066 69 16.75 ± 0.205.294 ± 0.064 32 16.96 ± 0.20 5.227 ± 0.062 53 19.95 ± 0.20 4.450 ±0.045 39 20.22 ± 0.20 4.391 ± 0.043 20 23.18 ± 0.20 3.837 ± 0.033 3823.92 ± 0.20 3.721 ± 0.031 41 24.40 ± 0.20 3.648 ± 0.030 44 24.73 ± 0.203.600 ± 0.029 22 24.99 ± 0.20 3.564 ± 0.028 50 25.12 ± 0.20 3.545 ±0.028 28 25.39 ± 0.20 3.509 ± 0.027 51 25.70 ± 0.20 3.466 ± 0.027 2126.19 ± 0.20 3.403 ± 0.026 27 26.72 ± 0.20 3.336 ± 0.025 30 27.02 ± 0.203.300 ± 0.024 25 27.34 ± 0.20 3.262 ± 0.024 23 28.44 ± 0.20 3.138 ±0.022 20

In some embodiments, the free base of crystalline Compound 1 comprisesthe crystalline Form II. In some preferred embodiments, the free base ofcrystalline Compound 1 comprises the crystalline Form II and less than25 mole %, 10 mole % or 5 mole % of crystalline Form I, crystallineMaterial N or amorphous forms of Compound 1.

In a preferred embodiment, the crystalline Form II is prepared from aslurry comprising the free base of Compound 1 in heptane, from which thecrystalline Form II is formed and filtered. Thus, in some embodiments,the crystalline Form II comprises residual (1-500 ppm) heptane. Inanother preferred embodiment, the crystalline Form II is prepared from aslurry comprising the free base of Compound 1 in water, from which thecrystalline Form II is formed and filtered.

There are several advantages of crystalline Form II relative tocrystalline Form I or Material N. For example, the crystalline Form IIcan be prepared from a slurry comprising the free base of Compound 1 inheptane, which is suitable for good manufacturing practices (GMP)protocols. Further, in a most preferred embodiment, the crystalline FormII can be prepared from a slurry comprising the free base of Compound 1in water or the HCl salt of Compound 1 in water, thus reducing oreliminating the need for solvent during recrystalization. Thus, in someembodiments, crystalline Form II of Compound 1 comprises less than 500ppm, 100 ppm, less than 50 ppm or less than 10 ppm organic solvent.Also, Form II has less of a propensity than Material N to agglomerateupon size reduction, e.g., upon milling. As such, Form II has greaterflowability than Material N. Certain illustrative and non-limitingadvantages of Form II over Material N (i.e., Form N) are shown in thetable below.

DATA/ EXPERIMENT RESULTS/STATUS Identify Form N: suitable Limited numberof suitable solvents compared to Form II solvent for MTBE identified(suitable for GMP; Class III solvent) scale-up Scale-up results lookgood Form II: More solvent options than Form N, including H₂O Currentsolvent is heptane (suitable for GMP; Class III solvent) produced on 5kg scale Formation time faster than N (could translate to 2-3 day savingin production time) Better recovery than N Size/ Acicular morphologyobserved for form N; material Morphology composed of small and largeparticles of N and II Agglomerates are an issue for Form N relative toForm II (less agglomeration seen with energy-reduced method) PK Oraladministrations of GBT440 Forms N and II to rats Comparison resulted incomparable exposure at 100 & 500 mg/kg of N and II

Material N

In another embodiment, the crystalline Compound 1 free base comprisescrystalline Material N, which is characterized by an endothermic peak at(95±2) C as measured by differential scanning calorimetry. The terms“Material N”, “form N” and “polymorphic form N” are used interchangeablyherein. In another embodiment, the crystalline Material N of the freebase of crystalline Compound 1 is characterized by the substantialabsence of thermal events at temperatures below the endothermic peak at(95±2) ° C. as measured by differential scanning calorimetry. In anotherembodiment, the crystalline Material N of the free base of crystallineCompound 1 is characterized by an X-ray powder diffraction peak (Cu Kαradiation at one or more of 11.65°, 11.85°, 12.08°, 16.70°, 19.65° or23.48 °2θ. In another embodiment, the crystalline Material N of the freebase of crystalline Compound 1 is characterized by an X-ray powderdiffraction pattern (Cu Kα radiation) substantially similar to that ofFIG. 7.

In another embodiment, the crystalline Material N of the free base ofcrystalline Compound 1 is characterized by at least one X-ray powderdiffraction peak (Cu Kα radiation) selected from 11.65°, 11.85°, 12.08°,16.70°, 19.65° and 23.48 °2θ (each ±0.2° 2θ). In another embodiment, thecrystalline Material N of the free base of crystalline Compound 1 ischaracterized by at least two X-ray powder diffraction peaks (Cu Kαradiation) selected from 11.65°, 11.85°, 12.08°, 16.70°, 19.65° and23.48 °2θ (each ±0.2 °2θ). In another embodiment, the crystallineMaterial N of the free base of crystalline Compound 1 is characterizedby at least three X-ray powder diffraction peaks (Cu Kα radiation)selected from 11.65°, 11.85°, 12.08°, 16.70°, 19.65° and 23.48 °2θ (each±0.2 °2θ).

In another embodiment, Material N is characterized by 1, 2, 3, 4, ormore peaks as tabulated below.

Observed Peaks for Material N, XRPD File 615765.

°2θ d space (Å) Intensity (%)  5.55 ± 0.20 15.924 ± 0.595  54 11.65 ±0.20 7.597 ± 0.132 31 11.85 ± 0.20 7.468 ± 0.128 50 12.08 ± 0.20 7.324 ±0.123 31 12.67 ± 0.20 6.987 ± 0.112 29 13.12 ± 0.20 6.748 ± 0.104 8314.94 ± 0.20 5.929 ± 0.080 34 15.19 ± 0.20 5.832 ± 0.077 56 15.76 ± 0.205.623 ± 0.072 20 16.70 ± 0.20 5.310 ± 0.064 100 17.35 ± 0.20 5.112 ±0.059 52 19.65 ± 0.20 4.517 ± 0.046 60 23.48 ± 0.20 3.789 ± 0.032 7223.68 ± 0.20 3.757 ± 0.032 29 25.25 ± 0.20 3.527 ± 0.028 20 25.47 ± 0.203.497 ± 0.027 20 25.70 ± 0.20 3.466 ± 0.027 85 26.04 ± 0.20 3.422 ±0.026 35 26.37 ± 0.20 3.380 ± 0.025 55

In some embodiments, the free base of crystalline Compound 1 comprisesthe crystalline Material N and less than 25 mole %, 10 mole % or 5 mole% of crystalline Forms I or II or amorphous forms of Compound 1.

In another embodiment, the crystalline Material N is prepared from aslurry comprising the free base of Compound 1 in methyl tertiary butylether (MTBE), from which the crystalline—Material N is formed andfiltered. Thus, in some embodiments, the crystalline Material Ncomprises residual (1-500 ppm) MTBE.

There are several advantages of crystalline Material N relative tocrystalline Forms I or II. For example, the crystalline Material N canbe prepared from a slurry comprising the free base of Compound 1 inMTBE, which is suitable for good manufacturing practices (GMP)protocols.

In some embodiments, the crystalline ansolvate forms are stable tocontact with water, heptane, iso propyl ether (IPE), MTBE, and toluene,and such other solvents.

In another of its composition embodiments, this invention provides for apharmaceutical composition comprising a pharmaceutically acceptableexcipient and a crystalline Compound 1 free base, comprising one or moreof Form I, Form II or Material N.

In one of its method embodiments, this invention provides a method ofpreparing the solid crystalline free base of Compound 1 comprising,e.g., Form I, Form II and/or Material N.

In yet another of its method embodiments, there are provided methods forincreasing oxygen affinity of hemoglobin S in a subject, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a crystalline free base of Compound 1, comprising,e.g., Form I, Form II and/or Material N.

In yet another of its method embodiments, there are provided methods fortreating oxygen deficiency associated with sickle cell anemia in asubject, the method comprising administering to a subject in needthereof a therapeutically effective amount of a crystalline free base ofCompound 1, comprising, e.g., Form I, Form II and/or Material N.

In all of such treatments, the effective amount of free base of Compound1, comprising e.g., Form I, Form II and/or Material N to the treatedpatient is already disclosed in the art.

Solvates

This invention arises in part out of the discovery that ansolvatepolymorphs of the free base of Compound 1 form solvate polymorphs with avariety of solvents, preferably other than certain hydrocarbon solvents,water and ethers.

Solvates of the crystalline free base of Compound 1 (e.g., from acetone,acetonitrile, dichloromethane, dioxane, ethanol, ethyl acetate,isopropyl alcohol, methyl ethyl ketone (MEK) and tetrahydrofuran) arealso contemplated to be used e.g., as intermediates to regenerate thefree base crystalline ansolvate of Compound 1. Such methods can include,without limitation, subjecting the solvate to vacuum conditions; and/orgenerating a salt and disproportionating it in water to form theansolvate; and/or slurrying or washing the solvate with a solvent lessprone to solvate formation such as heptane, di-isopropyl ether (IPE),tert-methyl butyl ether (MTBE) and toluene.

In another of its composition embodiments, this invention provides for apharmaceutical composition comprising a pharmaceutically acceptableexcipient and one or more of the solvated crystal forms provided herein.

In one of its method embodiments, this invention provides a method ofpreparing the solvated crystal forms provided herein.

In yet another of its method embodiments, there are provided methods forincreasing oxygen affinity of hemoglobin S in a subject, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of one or more of the solvated crystal forms providedherein.

In yet another of its method embodiments, there are provided methods fortreating oxygen deficiency associated with sickle cell anemia in asubject, the method comprising administering to a subject in needthereof a therapeutically effective amount of one or more of thesolvated crystal forms provided herein.

In all of such treatments, the effective amount of the free base ofCompound 1, to the treated patient is already disclosed in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a XRPD profile of the crystalline HCl salt before (top) andafter (bottom) 5 minutes slurried in water.

FIG. 2 is a XRPD profile of the free base Form I (top), Form II(middle), and Material N (bottom).

FIG. 3 is a XRPD profile and contemplated indexing for free base Form I.

FIG. 4 is a thermal characterization for free base Form I.

FIG. 5 is a XRPD profile and contemplated indexing for free base FormII.

FIG. 6 is a thermal characterization for free base Form II.

FIG. 7 is a XRPD profile for free base Material N.

FIG. 8 is a thermal characterization for free base Material N.

FIG. 9 depicts an Energy-Temperature Diagram between the Free Base FormsI, II, and Material N. The enthalpy (H) and free energy (G) isobars foreach form are depicted as a function of temperature. ΔH_(f) is the heatof fusion; T is transition temperature; m is melt temperature;superscripts I, II, and N refer to the polymorphs. *Under the testconditions, not enough information was available to graphicallyrepresent the free energy isobar of Form I below 6° C. and above theestimated transition temperature T^(N-II); the isobar likely intersectsG_(L) at a temperature below m^(II), allowing the possibility that FormI may be enantiotropic with Form II (where T^(I-II) occurs below 6° C.)and/or Material N (where either T^(I-N) occurs below T^(I-II) or T^(N-I)occurs above T^(N-II), but not both). Free energy isobars can onlyintersect each other once.

FIG. 10 depicts ¹³C Solid State NMR spectra for Free Base Forms I(bottom), II (middle), and Material N (top). Form I contains onemolecule per asymmetric unit. Material N contains four molecules perasymmetric unit. As observed by ¹³C Solid State NMR spectra, Forms IIand N did not undergo a transition over 250 K to 340 K. Chemical shiftschange slightly with temperature (not illustrated graphically).

FIG. 11 depicts ¹⁵N Solid State NMR spectra for Free Base Forms I(bottom), II (middle), and Material N (top).

FIG. 12 depicts a differential scanning calorimetry (DSC) curve for FreeBase Material N.

FIG. 13 depicts a DSC curve for Free Base Form II.

FIG. 14 depicts a DSC curve for Free Base Form I.

FIG. 15 depicts a XRPD profile of maturation experiments for the freebase of Compound 1 at multiple temperatures.

FIG. 16 depicts a contemplated XRPD profile for solvated Material E.

FIG. 17 depicts a contemplated XRPD profile for solvated Material F.

FIG. 18 depicts a contemplated XRPD profile for solvated Material G.

FIG. 19 depicts a contemplated XRPD profile for solvated Material H.

FIG. 20 depicts a contemplated XRPD profile for solvated Material J.

FIG. 21 depicts a contemplated XRPD profile for solvated Material K.

FIG. 22 depicts a contemplated XRPD profile for solvated Material L.

FIG. 23 depicts a contemplated XRPD profile for solvated Material M.

FIG. 24 depicts a contemplated XRPD profile for solvated Material O.

FIG. 25 depicts an XRPD profile comparison of contemplated isostructuralsolvates of the free base of Compound 1. From top to bottom: Material Efrom acetone; Material F from ACN; Material G from DCM; Material H fromdioxane; Material J from EtOH; Material K from IPA/water (also obtainedfrom IPA); and Material L from THF, Material M from MEK.

DETAILED DESCRIPTION

As noted above, this invention is directed, in part, to a stable freebase of Compound 1 and, in particular, the free base Form I, Form II orMaterial N. However, prior to discussing this invention in furtherdetail, the following terms will be defined.

Definitions

As used herein, the following terms have the following meanings.

The singular forms “a,” “an,” and “the” and the like include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a compound” includes both a single compound and aplurality of different compounds.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, and concentration, including a range,indicates approximations which may vary by ±10%, ±5% or ±1%.

“Administration” refers to introducing an agent into a patient. Atherapeutic amount can be administered, which can be determined by thetreating physician or the like. An oral route of administration ispreferred. The related terms and phrases administering” and“administration of”, when used in connection with a compound orpharmaceutical composition (and grammatical equivalents) refer both todirect administration, which may be administration to a patient by amedical professional or by self-administration by the patient, and/or toindirect administration, which may be the act of prescribing a drug. Forexample, a physician who instructs a patient to self-administer a drugand/or provides a patient with a prescription for a drug isadministering the drug to the patient. In any event, administrationentails delivery to the patient of the drug.

The “crystalline ansolvate” of Compound 1 is a crystalline solid form ofthe free base of2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde,such as, e.g., crystalline Form I, Form II or Material N as disclosedherein. Each of the Form I, Form II or Material N crystal lattices issubstantially free of solvents of crystallization. However, any solventpresent is not included in the crystal lattice and is randomlydistributed outside the crystal lattice. Therefore, Form I, Form II orMaterial N crystals in bulk may contain, outside the crystal lattice,small amounts of one or more solvents, such as the solvents used in itssynthesis or crystallization. As used above, “substantially free of” and“small amounts,” refers to the presence of solvents preferably less that10,000 parts per million (ppm), or more preferably, less than 500 ppm.

The “crystalline solvate” of Compound 1 is a crystalline solid form ofthe free base of2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde,where the crystal lattices comprises one or more solvents ofcrystallization.

“Characterization” refers to obtaining data which may be used toidentify a solid form of a compound, for example, to identify whetherthe solid form is amorphous or crystalline and whether it is unsolvatedor solvated. The process by which solid forms are characterized involvesanalyzing data collected on the polymorphic forms so as to allow one ofordinary skill in the art to distinguish one solid form from other solidforms containing the same material. Chemical identity of solid forms canoften be determined with solution-state techniques such as ¹³C NMR or ¹HNMR. While these may help identify a material, and a solvent moleculefor a solvate, such solution-state techniques themselves may not provideinformation about the solid state. There are, however, solid-stateanalytical techniques that can be used to provide information aboutsolid-state structure and differentiate among polymorphic solid forms,such as single crystal X-ray diffraction, X-ray powder diffraction(XRPD), solid state nuclear magnetic resonance (SS-NMR), and infraredand Raman spectroscopy, and thermal techniques such as differentialscanning calorimetry (DSC), Solid state ¹³C-NMR, thermogravimetry (TG),melting point, and hot stage microscopy.

To “characterize” a solid form of a compound, one may, for example,collect XRPD data on solid forms of the compound and compare the XRPDpeaks of the forms. For example, when only three solid forms, e.g.,Forms I and II and Material N, are compared and the Form I pattern showsa peak at an angle where no peaks appear in the Form II or Material Npattern, then that peak, for that compound, distinguishes Form I fromForm II and Material N and further acts to characterize Form I. Thecollection of peaks which distinguish e.g., Form I from the other knownforms is a collection of peaks which may be used to characterize Form I.Those of ordinary skill in the art will recognize that there are oftenmultiple ways, including multiple ways using the same analyticaltechnique, to characterize solid forms. Additional peaks could also beused, but are not necessary, to characterize the form up to andincluding an entire diffraction pattern. Although all the peaks withinan entire XRPD pattern may be used to characterize such a form, a subsetof that data may, and typically is, used to characterize the form.

An XRPD pattern is an x-y graph with diffraction angle (typically ° 2θ)on the x-axis and intensity on the y-axis. The peaks within this patternmay be used to characterize a crystalline solid form. As with any datameasurement, there is variability in XRPD data. The data are oftenrepresented solely by the diffraction angle of the peaks rather thanincluding the intensity of the peaks because peak intensity can beparticularly sensitive to sample preparation (for example, particlesize, moisture content, solvent content, and preferred orientationeffects influence the sensitivity), so samples of the same materialprepared under different conditions may yield slightly differentpatterns; this variability is usually greater than the variability indiffraction angles. Diffraction angle variability may also be sensitiveto sample preparation. Other sources of variability come from instrumentparameters and processing of the raw X-ray data: different X-rayinstruments operate using different parameters and these may lead toslightly different XRPD patterns from the same solid form, and similarlydifferent software packages process X-ray data differently and this alsoleads to variability. These and other sources of variability are knownto those of ordinary skill in the pharmaceutical arts. Due to suchsources of variability, it is usual to assign a variability of ±0.2° 2θto diffraction angles in XRPD patterns.

“Comprising” or “comprises” is intended to mean that the compositionsand methods include the recited elements, but not exclude others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination for the stated purpose. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

Form II and Material N are enantiotropic at a transition temperature (ofapproximately 42° C.). Below this transition temperature, Material N ofthe free base of Compound 1 is the thermodynamically more stable formrelative to Forms I and II. Above this transition temperature, Form IIof the free base of Compound 1 is the thermodynamically more stable formrelative to Form I and Material N.

“Room temperature” refers to (22±5) ° C.

“Therapeutically effective amount” or “therapeutic amount” refers to anamount of a drug or an agent that when administered to a patientsuffering from a condition, will have the intended therapeutic effect,e.g., alleviation, amelioration, palliation or elimination of one ormore manifestations of the condition in the patient. The therapeuticallyeffective amount will vary depending upon the subject and the conditionbeing treated, the weight and age of the subject, the severity of thecondition, the particular composition or excipient chosen, the dosingregimen to be followed, timing of administration, the manner ofadministration and the like, all of which can be determined readily byone of ordinary skill in the art. The full therapeutic effect does notnecessarily occur by administration of one dose, and may occur onlyafter administration of a series of doses. Thus, a therapeuticallyeffective amount may be administered in one or more administrations. Forexample, and without limitation, a therapeutically effective amount ofan agent, in the context of treating disorders related to hemoglobin S,refers to an amount of the agent that alleviates, ameliorates,palliates, or eliminates one or more manifestations of the disordersrelated to hemoglobin S in the patient.

“Treatment”, “treating”, and “treat” are defined as acting upon adisease, disorder, or condition with an agent to reduce or amelioratethe harmful or any other undesired effects of the disease, disorder, orcondition and/or its symptoms. Treatment, as used herein, covers thetreatment of a human patient, and includes: (a) reducing the risk ofoccurrence of the condition in a patient determined to be predisposed tothe disease but not yet diagnosed as having the condition, (b) impedingthe development of the condition, and/or (c) relieving the condition,i.e., causing regression of the condition and/or relieving one or moresymptoms of the condition. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, multilineagehematologic improvement, decrease in the number of required bloodtransfusions, decrease in infections, decreased bleeding, and the like.

Identifying Forms I, II and Material N

When the HCl salt of Compound 1 was subjected to various stressconditions, disproportionation of the HCl salt in water was observed togenerate the free base. At least three anhydrous crystalline forms ofthe free base were identified, termed Free Base Forms I, II, andMaterial N. It was discovered that nucleation of Free Base Form Igenerally occurs first and that extending the slurry time induces thetransformation of Free Base Form I to Free Base Form II, a morethermodynamically stable phase relative to Form I. It was furtherdiscovered that Free Base Material N appears to be most stable form,relative to Forms I and II, at room temperature. Free Base Material Nwas found to be enantiotropically active relative to Form II, and willtransform reversibly at a specific transition temperature (estimatedherein near 42° C.). Above the transition temperature, Free Base Form IIappears to be the most stable form, relative to Form I and Material N.

Based in part on solid-state nuclear magnetic resonance data, all threeforms are crystalline and are distinct polymorphic forms. See FIGS. 10and 11. Form I contains one molecule per asymmetric unit, Form IIcontains two molecules per asymmetric unit and Form N contains fourmolecules per asymmetric unit. See the ¹⁵N spectra in FIG. 11.

Ansolvates of Forms I, II and Material N

In one embodiment, this invention provides the free base crystallineansolvate of Compound 1. The free base crystalline ansolvate of Compound1 may include one or more of Form I, Form II and/or Material Npolymorphs. In some embodiments, the free base crystalline ansolvate ofCompound 1 may include the Form II polymorph. Preferably, the free basecrystalline ansolvate of Compound 1 may include Form II and/or MaterialN polymorphs. More preferably, the free base crystalline ansolvate ofCompound 1 may include the Material N polymorph. Yet more preferably,the free base crystalline ansolvate of Compound 1 is substantially freeof a solvated polymorph of Compound 1 free base. Further yet morepreferably, the free base crystalline ansolvate of Compound 1 issubstantially free of other ansolvate polymorphs of Compound 1 freebase. “Substantially free” of a component as used herein refers tocontain up to about 5%, more preferably about 3%, and still morepreferably about 1% of that component. As used herein, solvate includesa hydrate form as well.

Solvates of Compound 1

In one aspect, provided is a crystalline solvate of Compound 1:

In some embodiments, the crystalline solvate is substantially free of anansolvated polymorph of Compound 1.

Many of the solubility and screen experiments with the free base ofCompound 1 resulted in precipitation of solids characterized as solvateformation with some solvents. Under the conditions, solvates were notobserved from the free base of Compound 1 with four solvents, includingheptane, di-isopropyl ether (IPE), tert-methyl butyl ether (MTBE) andtoluene. Solvates were observed from the free base of Compound 1 in ninesolvents including acetone (Material E), acetonitrile (Material F),dichloromethane (Material G), dioxane (Material H), ethanol (MaterialJ), isopropyl alcohol or a mixture of water and isopropyl alcohol(Material K), tetrahydrofuran (Material L), methyl ethyl ketone “MEK”(Material M), ethyl acetate (Material O) and dimethyl sulfoxide “DMSO”(Material P). The majority of the solvates (i.e., Materials E-H, J-M, Oand P are contemplated to be isostructural. In some embodiments, thecrystalline solvate includes one or more of Material E, Material F,Material G, Material H, Material J, Material K, Material L, Material M,Material O or Material P.

Material E can be characterized by at least one X-ray powder diffractionpeak (Cu Kα radiation) selected from 8.69, 11.73, 12.10, 15.26, 16.11,17.45, 22.39, 22.55 and 23.70±0.20. Material F can be characterized byat least one X-ray powder diffraction peak (Cu Kα radiation) selectedfrom 8.47, 8.81, 12.75, 13.17, 14.92, 15.63, 17.01 23.73, and24.07±0.20. Material G can be characterized by at least one X-ray powderdiffraction peak (Cu Kα radiation) selected from 8.47, 11.45, 12.62,14.66, 15.69, 17.01, 18.47, 20.32, 22.61, 23.08, 23.43 and 23.70±0.20.Material H can be characterized by at least one X-ray powder diffractionpeak (Cu Kα radiation) selected from 8.61, 11.67, 15.33, 16.28, 17.28,22.58, 23.51 and 25.77±0.20. Material J can be characterized by at leastone X-ray powder diffraction peak (Cu Kα radiation) selected from 8.52,8.88, 12.79, 15.04, 15.61, 17.11, 22.81, 23.87, 24.17, 24.62 and26.44±0.20. Material K can be characterized by at least one X-ray powderdiffraction peak (Cu Kα radiation) selected from 8.52; 8.83, 11.35,15.04, 15.74, 17.11, 23.46, 23.58, 24.08 and 25.99±0.20. Material L canbe characterized by at least one X-ray powder diffraction peak (Cu Kαradiation) selected from 8.61, 8.78, 11.67, 14.94, 15.28, 16.14, 17.30,22.75, 23.71 and 26.05±0.20; and Material M can be characterized by atleast one X-ray powder diffraction peak (Cu Kα radiation) selected from7.74, 10.05, 12.82, 15.33, 16.80, 20.82, 21.14, 25.80 and 26.97±0.20.

The solvates (such as, of acetone, acetonitrile, dichloromethane,dioxane, ethanol, ethyl acetate, isopropyl alcohol, MEK, tetrahydrofuranor DMSO) could be used e.g., as intermediates to regenerate the freebase crystalline ansolvate of Compound 1 by several methods includingsubjecting the solvate to vacuum conditions; and/or regenerating the HClsalt and disproportionating HCl; and/or washing the solvate with asolvent less prone to solvate formation such as heptane, di-isopropylether (IPE), tert-methyl butyl ether (MTBE) and toluene.

TABLE 1 Data Related to Solvates of the Free Base of Compound 1Estimated Number of Volume per Crystal- Formula Formula lization VolumeUnits Unit* Indexing Identifier Solvent (Å³/Cell) per Cell (Å³) ResultMaterial E acetone 968 2 484 FIG. 1 Material F ACN 947 2 473 FIG. 2Material G DCM 959 2 480 FIG. 3 Material H dioxane 977 2 488 FIG. 4Material J EtOH 943 2 472 FIG. 5 Material K IPA 963 2 481 FIG. 6Material L THF 972 2 486 FIG. 7 Material M MEK 3956 8 494 FIG. 8Material O EtOAc — — — FIG. 9 Material P** DMSO — — — — *The value isobtained by dividing the volume of the cell, derived from the tentativeindexing solution, by the number of formula units within the cell.**Material P was observed as a mixture with a “sulfate form I”.

Certain contemplated peaks of the various solvates provided herein aretabulated below. Certain peaks, which are preferably non-overlapping,low-angle peaks, with strong intensity, were not identified. The peakswere determined to the extent that the state of preferred orientation inthe samples were unknown.

TABLE 2 Observed peaks for Material E. °2θ d space (Å) Intensity (%) 8.41 ± 0.20 10.517 ± 0.256  13  8.69 ± 0.20 10.174 ± 0.239  100 11.73 ±0.20 7.543 ± 0.130 17 12.10 ± 0.20 7.314 ± 0.122 20 13.00 ± 0.20 6.809 ±0.106 15 14.02 ± 0.20 6.316 ± 0.091 5 14.77 ± 0.20 5.996 ± 0.082 1615.26 ± 0.20 5.807 ± 0.077 34 15.81 ± 0.20 5.605 ± 0.071 7 16.11 ± 0.205.501 ± 0.069 20 16.48 ± 0.20 5.379 ± 0.066 11 16.65 ± 0.20 5.326 ±0.064 11 16.88 ± 0.20 5.253 ± 0.063 3 17.26 ± 0.20 5.136 ± 0.060 9 17.45± 0.20 5.083 ± 0.058 32 20.02 ± 0.20 4.435 ± 0.044 2 20.92 ± 0.20 4.246± 0.041 13 21.91 ± 0.20 4.057 ± 0.037 20 22.39 ± 0.20 3.970 ± 0.035 4922.55 ± 0.20 3.944 ± 0.035 37 22.81 ± 0.20 3.898 ± 0.034 16 23.36 ± 0.203.807 ± 0.032 12 23.70 ± 0.20 3.755 ± 0.032 61 24.37 ± 0.20 3.653 ±0.030 12 24.85 ± 0.20 3.583 ± 0.029 5 25.42 ± 0.20 3.504 ± 0.027 2 25.89± 0.20 3.442 ± 0.026 8 26.19 ± 0.20 3.403 ± 0.026 40 26.97 ± 0.20 3.306± 0.024 3 27.61 ± 0.20 3.231 ± 0.023 16 28.24 ± 0.20 3.160 ± 0.022 228.48 ± 0.20 3.134 ± 0.022 5 28.69 ± 0.20 3.111 ± 0.021 7 29.83 ± 0.202.995 ± 0.020 4

TABLE 3 Observed peaks for Material F. °2θ d space (Å) Intensity (%) 8.47 ± 0.20 10.434 ± 0.252  100  8.81 ± 0.20 10.039 ± 0.233  49 11.42 ±0.20 7.752 ± 0.138 15 12.75 ± 0.20 6.942 ± 0.110 27 13.17 ± 0.20 6.723 ±0.103 21 13.87 ± 0.20 6.384 ± 0.093 7 14.61 ± 0.20 6.064 ± 0.084 1314.92 ± 0.20 5.936 ± 0.080 43 15.51 ± 0.20 5.713 ± 0.074 24 15.63 ± 0.205.671 ± 0.073 43 15.96 ± 0.20 5.553 ± 0.070 15 17.01 ± 0.20 5.212 ±0.062 31 17.26 ± 0.20 5.136 ± 0.060 4 17.70 ± 0.20 5.011 ± 0.057 9 18.17± 0.20 4.883 ± 0.054 4 18.79 ± 0.20 4.724 ± 0.050 10 19.35 ± 0.20 4.587± 0.047 4 19.49 ± 0.20 4.555 ± 0.047 3 20.02 ± 0.20 4.435 ± 0.044 420.29 ± 0.20 4.377 ± 0.043 9 21.06 ± 0.20 4.219 ± 0.040 11 21.33 ± 0.204.167 ± 0.039 4 22.71 ± 0.20 3.915 ± 0.034 27 23.11 ± 0.20 3.848 ± 0.03315 23.73 ± 0.20 3.749 ± 0.031 42 24.07 ± 0.20 3.698 ± 0.031 59 24.65 ±0.20 3.612 ± 0.029 87 24.95 ± 0.20 3.569 ± 0.028 6 25.20 ± 0.20 3.534 ±0.028 5 25.69 ± 0.20 3.468 ± 0.027 15 26.52 ± 0.20 3.361 ± 0.025 6126.79 ± 0.20 3.328 ± 0.025 10 27.02 ± 0.20 3.300 ± 0.024 9

TABLE 4 Observed peaks for Material G. °2θ d space (Å) Intensity (%) 8.47 ± 0.20 10.434 ± 0.252  45  8.76 ± 0.20 10.096 ± 0.235  12 11.45 ±0.20 7.729 ± 0.137 76 12.62 ± 0.20 7.015 ± 0.113 36 13.09 ± 0.20 6.765 ±0.105 10 13.87 ± 0.20 6.384 ± 0.093 5 14.66 ± 0.20 6.044 ± 0.083 3914.92 ± 0.20 5.936 ± 0.080 26 15.33 ± 0.20 5.782 ± 0.076 7 15.69 ± 0.205.647 ± 0.072 88 16.01 ± 0.20 5.536 ± 0.070 8 16.76 ± 0.20 5.289 ± 0.06315 17.01 ± 0.20 5.212 ± 0.062 29 17.50 ± 0.20 5.068 ± 0.058 5 17.60 ±0.20 5.040 ± 0.057 4 18.13 ± 0.20 4.892 ± 0.054 5 18.47 ± 0.20 4.804 ±0.052 21 19.55 ± 0.20 4.540 ± 0.046 4 20.01 ± 0.20 4.439 ± 0.044 5 20.32± 0.20 4.370 ± 0.043 20 21.11 ± 0.20 4.209 ± 0.040 15 22.61 ± 0.20 3.932± 0.035 42 22.88 ± 0.20 3.887 ± 0.034 9 23.08 ± 0.20 3.854 ± 0.033 2823.43 ± 0.20 3.797 ± 0.032 56 23.70 ± 0.20 3.755 ± 0.032 48 24.12 ± 0.203.690 ± 0.030 13 24.42 ± 0.20 3.646 ± 0.030 100 25.05 ± 0.20 3.555 ±0.028 7 25.40 ± 0.20 3.506 ± 0.027 26 26.36 ± 0.20 3.382 ± 0.025 5026.57 ± 0.20 3.355 ± 0.025 7 26.82 ± 0.20 3.324 ± 0.025 27 27.07 ± 0.203.294 ± 0.024 10

TABLE 5 Observed peaks for Material H. °2θ d space (Å) Intensity (%) 8.61 ± 0.20 10.273 ± 0.244  48  8.81 ± 0.20 10.039 ± 0.233  20 11.67 ±0.20 7.586 ± 0.132 32 12.10 ± 0.20 7.314 ± 0.122 11 12.79 ± 0.20 6.924 ±0.110 9 14.56 ± 0.20 6.085 ± 0.084 4 14.87 ± 0.20 5.956 ± 0.081 22 15.33± 0.20 5.782 ± 0.076 42 15.76 ± 0.20 5.623 ± 0.072 18 16.28 ± 0.20 5.445± 0.067 51 16.73 ± 0.20 5.299 ± 0.064 9 17.28 ± 0.20 5.132 ± 0.060 6117.68 ± 0.20 5.016 ± 0.057 3 20.47 ± 0.20 4.338 ± 0.042 12 21.38 ± 0.204.157 ± 0.039 7 21.83 ± 0.20 4.072 ± 0.037 4 22.23 ± 0.20 3.999 ± 0.0369 22.58 ± 0.20 3.938 ± 0.035 100 22.95 ± 0.20 3.876 ± 0.034 6 23.11 ±0.20 3.848 ± 0.033 14 23.51 ± 0.20 3.783 ± 0.032 88 24.37 ± 0.20 3.653 ±0.030 13 24.65 ± 0.20 3.612 ± 0.029 10 25.77 ± 0.20 3.457 ± 0.027 4126.67 ± 0.20 3.342 ± 0.025 7 26.97 ± 0.20 3.306 ± 0.024 5 27.66 ± 0.203.225 ± 0.023 3 28.11 ± 0.20 3.174 ± 0.022 4 28.61 ± 0.20 3.120 ± 0.0226 28.96 ± 0.20 3.083 ± 0.021 4 29.23 ± 0.20 3.055 ± 0.021 3 29.63 ± 0.203.015 ± 0.020 3

TABLE 6 Observed peaks for Material J. °2θ d space (Å) Intensity (%) 8.52 ± 0.20 10.373 ± 0.249  100  8.88 ± 0.20 9.964 ± 0.229 39 11.33 ±0.20 7.809 ± 0.140 22 12.79 ± 0.20 6.924 ± 0.110 25 13.12 ± 0.20 6.748 ±0.104 24 13.94 ± 0.20 6.354 ± 0.092 4 14.47 ± 0.20 6.120 ± 0.085 1415.04 ± 0.20 5.890 ± 0.079 42 15.61 ± 0.20 5.677 ± 0.073 56 15.84 ± 0.205.594 ± 0.071 16 17.11 ± 0.20 5.181 ± 0.061 33 17.40 ± 0.20 5.097 ±0.059 4 17.82 ± 0.20 4.979 ± 0.056 8 18.12 ± 0.20 4.897 ± 0.054 3 18.90± 0.20 4.695 ± 0.050 11 19.39 ± 0.20 4.579 ± 0.047 5 19.62 ± 0.20 4.525± 0.046 4 20.16 ± 0.20 4.406 ± 0.044 8 20.96 ± 0.20 4.239 ± 0.040 1222.81 ± 0.20 3.898 ± 0.034 27 23.15 ± 0.20 3.843 ± 0.033 9 23.28 ± 0.203.821 ± 0.033 7 23.87 ± 0.20 3.729 ± 0.031 34 24.17 ± 0.20 3.683 ± 0.03052 24.62 ± 0.20 3.616 ± 0.029 95 25.20 ± 0.20 3.534 ± 0.028 5 25.77 ±0.20 3.457 ± 0.027 13 26.44 ± 0.20 3.371 ± 0.025 70 26.71 ± 0.20 3.338 ±0.025 10 27.21 ± 0.20 3.278 ± 0.024 7

TABLE 7 Observed peaks for GBT000440, Material K. °2θ d space (Å)Intensity (%)  8.52 ± 0.20 10.373 ± 0.249  75  8.83 ± 0.20 10.020 ±0.232  33 11.35 ± 0.20 7.797 ± 0.139 29 12.52 ± 0.20 7.071 ± 0.114 2112.90 ± 0.20 6.861 ± 0.108 24 13.92 ± 0.20 6.361 ± 0.092 4 14.49 ± 0.206.113 ± 0.085 18 15.04 ± 0.20 5.890 ± 0.079 41 15.34 ± 0.20 5.775 ±0.076 17 15.74 ± 0.20 5.629 ± 0.072 57 15.93 ± 0.20 5.564 ± 0.070 1316.61 ± 0.20 5.336 ± 0.065 7 17.11 ± 0.20 5.181 ± 0.061 33 17.70 ± 0.205.011 ± 0.057 7 18.00 ± 0.20 4.928 ± 0.055 4 18.38 ± 0.20 4.826 ± 0.05313 19.04 ± 0.20 4.662 ± 0.049 4 19.74 ± 0.20 4.498 ± 0.046 5 20.21 ±0.20 4.395 ± 0.043 11 20.99 ± 0.20 4.232 ± 0.040 12 22.70 ± 0.20 3.918 ±0.034 22 22.90 ± 0.20 3.884 ± 0.034 17 23.46 ± 0.20 3.791 ± 0.032 4523.58 ± 0.20 3.773 ± 0.032 70 24.08 ± 0.20 3.695 ± 0.030 100 24.75 ±0.20 3.597 ± 0.029 6 25.19 ± 0.20 3.536 ± 0.028 21 25.99 ± 0.20 3.429 ±0.026 71 26.71 ± 0.20 3.338 ± 0.025 11 27.36 ± 0.20 3.260 ± 0.024 928.11 ± 0.20 3.174 ± 0.022 4 28.69 ± 0.20 3.111 ± 0.021 9

TABLE 8 Observed peaks for Material L. °2θ d space (Å) Intensity (%) 8.61 ± 0.20 10.273 ± 0.244  79  8.78 ± 0.20 10.077 ± 0.235  38 11.67 ±0.20 7.586 ± 0.132 35 12.17 ± 0.20 7.274 ± 0.121 19 12.94 ± 0.20 6.844 ±0.107 14 14.07 ± 0.20 6.293 ± 0.090 3 14.62 ± 0.20 6.057 ± 0.084 5 14.94± 0.20 5.929 ± 0.080 25 15.28 ± 0.20 5.800 ± 0.076 50 15.93 ± 0.20 5.564± 0.070 18 16.14 ± 0.20 5.490 ± 0.068 49 16.33 ± 0.20 5.429 ± 0.067 916.70 ± 0.20 5.310 ± 0.064 9 16.85 ± 0.20 5.263 ± 0.063 6 17.30 ± 0.205.127 ± 0.060 52 17.63 ± 0.20 5.030 ± 0.057 6 18.37 ± 0.20 4.830 ± 0.0533 20.14 ± 0.20 4.409 ± 0.044 5 20.59 ± 0.20 4.314 ± 0.042 14 21.53 ±0.20 4.128 ± 0.038 11 22.01 ± 0.20 4.038 ± 0.037 3 22.44 ± 0.20 3.961 ±0.035 27 22.75 ± 0.20 3.910 ± 0.034 72 23.10 ± 0.20 3.851 ± 0.033 2023.31 ± 0.20 3.816 ± 0.033 19 23.48 ± 0.20 3.789 ± 0.032 12 23.71 ± 0.203.752 ± 0.031 100 24.48 ± 0.20 3.636 ± 0.029 20 24.70 ± 0.20 3.604 ±0.029 4 24.93 ± 0.20 3.571 ± 0.028 3 25.59 ± 0.20 3.482 ± 0.027 5 25.72± 0.20 3.464 ± 0.027 5 26.05 ± 0.20 3.420 ± 0.026 62 26.59 ± 0.20 3.352± 0.025 6 27.14 ± 0.20 3.286 ± 0.024 8 27.83 ± 0.20 3.206 ± 0.023 828.38 ± 0.20 3.145 ± 0.022 3 28.78 ± 0.20 3.102 ± 0.021 8 29.05 ± 0.203.074 ± 0.021 4 29.36 ± 0.20 3.042 ± 0.020 3

TABLE 9 Observed peaks for Material M. °2θ d space (Å) Intensity (%) 7.74 ± 0.20 11.424 ± 0.303  100  8.34 ± 0.20 10.601 ± 0.260  4 10.05 ±0.20 8.806 ± 0.178 17 12.82 ± 0.20 6.906 ± 0.109 46 13.05 ± 0.20 6.783 ±0.105 4 14.17 ± 0.20 6.249 ± 0.089 2 14.54 ± 0.20 6.092 ± 0.085 6 14.99± 0.20 5.910 ± 0.079 16 15.33 ± 0.20 5.782 ± 0.076 47 15.53 ± 0.20 5.707± 0.074 21 16.80 ± 0.20 5.278 ± 0.063 27 18.33 ± 0.20 4.839 ± 0.053 319.17 ± 0.20 4.630 ± 0.048 22 20.19 ± 0.20 4.399 ± 0.044 23 20.82 ± 0.204.266 ± 0.041 32 21.14 ± 0.20 4.202 ± 0.040 27 21.29 ± 0.20 4.173 ±0.039 14 22.01 ± 0.20 4.038 ± 0.037 13 22.28 ± 0.20 3.991 ± 0.036 2322.93 ± 0.20 3.879 ± 0.034 6 23.35 ± 0.20 3.810 ± 0.032 11 24.00 ± 0.203.708 ± 0.031 14 24.25 ± 0.20 3.670 ± 0.030 3 24.88 ± 0.20 3.578 ± 0.02911 25.54 ± 0.20 3.488 ± 0.027 9 25.80 ± 0.20 3.453 ± 0.027 94 26.97 ±0.20 3.306 ± 0.024 27 27.63 ± 0.20 3.229 ± 0.023 2 28.41 ± 0.20 3.142 ±0.022 7 28.54 ± 0.20 3.127 ± 0.022 8 29.03 ± 0.20 3.076 ± 0.021 3 29.30± 0.20 3.049 ± 0.020 7 29.63 ± 0.20 3.015 ± 0.020 15

Pharmaceutical Compositions

In another of its composition embodiments, this invention provides for apharmaceutical composition comprising a pharmaceutically acceptableexcipient and crystalline free base ansolvate of Compound 1, preferablyincluding one or more of the Form I, Form II and/or Material Npolymorphs.

Such compositions can be formulated for different routes ofadministration. Although compositions suitable for oral delivery willprobably be used most frequently, other routes that may be used includeintravenous, intraarterial, pulmonary, rectal, nasal, vaginal, lingual,intramuscular, intraperitoneal, intracutaneous, intracranial,subcutaneous and transdermal routes. Suitable dosage forms foradministering any of the compounds described herein include tablets,capsules, pills, powders, aerosols, suppositories, parenterals, and oralliquids, including suspensions, solutions and emulsions. Sustainedrelease dosage forms may also be used, for example, in a transdermalpatch form. All dosage forms may be prepared using methods that arestandard in the art (see e.g., Remington's Pharmaceutical Sciences,16^(th) ed., A. Oslo editor, Easton Pa. 1980).

Pharmaceutically acceptable excipients are non-toxic, aidadministration, and do not adversely affect the therapeutic benefit ofthe compound of this invention. Such excipients may be any solid,liquid, semi-solid or, in the case of an aerosol composition, gaseousexcipient that is generally available to one of skill in the art.Pharmaceutical compositions in accordance with the invention areprepared by conventional means using methods known in the art.

The compositions disclosed herein may be used in conjunction with any ofthe vehicles and excipients commonly employed in pharmaceuticalpreparations, e.g., talc, gum arabic, lactose, starch, magnesiumstearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffinderivatives, glycols, etc. Coloring and flavoring agents may also beadded to preparations, particularly to those for oral administration.Solutions can be prepared using water or physiologically compatibleorganic solvents such as ethanol, 1,2-propylene glycol, polyglycols,dimethylsulfoxide, fatty alcohols, triglycerides, partial esters ofglycerin and the like.

Solid pharmaceutical excipients include starch, cellulose, hydroxypropylcellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk and the like. Liquid andsemisolid excipients may be selected from glycerol, propylene glycol,water, ethanol and various oils, including those of petroleum, animal,vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineraloil, sesame oil, etc. In certain embodiments, the compositions providedherein comprises one or more of α-tocopherol, gum arabic, and/orhydroxypropyl cellulose.

In one embodiment, this invention provides sustained releaseformulations such as drug depots or patches comprising an effectiveamount of a compound provided herein. In another embodiment, the patchfurther comprises gum Arabic or hydroxypropyl cellulose separately or incombination, in the presence of alpha-tocopherol. Preferably, thehydroxypropyl cellulose has an average MW of from 10,000 to 100,000. Ina more preferred embodiment, the hydroxypropyl cellulose has an averageMW of from 5,000 to 50,000.

Compounds and pharmaceutical compositions of this invention maybe usedalone or in combination with other compounds. When administered withanother agent, the co-administration can be in any manner in which thepharmacological effects of both are manifest in the patient at the sametime. Thus, co-administration does not require that a singlepharmaceutical composition, the same dosage form, or even the same routeof administration be used for administration of both the compound ofthis invention and the other agent or that the two agents beadministered at precisely the same time. However, co-administration willbe accomplished most conveniently by the same dosage form and the sameroute of administration, at substantially the same time. Obviously, suchadministration most advantageously proceeds by delivering both activeingredients simultaneously in a novel pharmaceutical composition inaccordance with the present invention.

Preparative and Treatment Methods Ansolvate

In another aspect, the present invention provides a method of preparingthe crystalline free base ansolvate of Compound 1. In one embodiment,provided herein is a method of preparing the crystalline free base ofCompound 1 comprising slurrying or contacting the HCl salt of theCompound 1 with water and allowing dissociation of HCl to produce thefree base of Compound 1. In one embodiment, the crystalline free baseansolvate of Compound 1 prepared comprises one or more of Form I, FormII and Material N.

In yet another of its method embodiments, there are provided methods forincreasing oxygen affinity of hemoglobin S in a subject, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of a crystalline free base of Compound 1. In someembodiments, the crystalline free base of Compound 1 is an ansolvate. Inone embodiment, the crystalline free base of Compound 1 comprises one ormore of Form I, Form II and Material N.

In yet another of its method embodiments, there are provided methods fortreating oxygen deficiency associated with sickle cell anemia in asubject, the method comprising administering to a subject in needthereof a therapeutically effective amount of a crystalline free base ofCompound 1. In some embodiments, the crystalline free base of Compound 1is an ansolvate. In one embodiment, the crystalline free base ofCompound 1 comprises one or more of Form I, Form II and Material N.

In further aspects of the invention, a method is provided for treatingsickle cell disease, the method comprising administering to a subject inneed thereof a therapeutically effective amount of a crystalline freebase of Compound 1. In some embodiments, the crystalline free base ofCompound 1 is an ansolvate. In one embodiment, the crystalline free baseof Compound 1 comprises one or more of Form I, Form II and Material N.In still further aspects of the invention, a method is provided fortreating cancer, a pulmonary disorder, stroke, high altitude sickness,an ulcer, a pressure sore, Alzheimer's disease, acute respiratorydisease syndrome, and a wound, the method comprising administering to asubject in need thereof a therapeutically effective amount of acrystalline free base of Compound 1. In some embodiments, thecrystalline free base of Compound 1 is an ansolvate. In one embodiment,the crystalline free base of Compound 1 comprises one or more of Form I,Form II and Material N.

In such treatments, the dosing of the crystalline free base of Compound1 to the treated patient is already disclosed in the art.

Solvates

In another aspect, the present invention provides a method of preparingthe crystalline free base solvates of Compound 1. In some embodiments, afree base ansolvate, as described herein (e.g, as obtained by slurryingan HCl salt of Compound 1 in water) of Compound 1 is contacted with asolvent as provided herein, including a mixture of solvents, to preparethe solvate. the solvent or the mixture of solvents. Thus, a solvent canbe a single solvent or substantially a single solvent or a mixture ofsolvents. When a mixture of solvents is used, a solvate can be producedhaving one or more of the individual constituent solvents of the solventmixture. In some embodiments, the solvent includes alcoholic solventssuch as mono di or higher alcohols or alkanols. In some embodiments, thesolvent includes chlorinated solvents such as dichloromethanechloroform, et cetera. In some embodiments, the solvent includes ketonesolvents such as alkanones and cycloalkanones. Certain solvents includewithout limitation, methanol, ethanol, 2-propanol, 2-methyl-1-propanol,1-butanol, acetonitrile, acetone, dichloromethane, dioxane, ortetrahydrofuran, or combinations thereof, optionally including water.

In another aspect, a method is provided for increasing oxygen affinityof hemoglobin S in a subject, the method comprising administering to asubject in need thereof a therapeutically effective amount of acrystalline solvate of Compound 1.

In another aspect, a method is provided for treating oxygen deficiencyassociated with sickle cell anemia, the method comprising administeringto a subject in need thereof a therapeutically effective amount of acrystalline solvate of Compound 1.

EXAMPLES

The following examples describe the preparation, characterization, andproperties of the free base of Compound 1 Form I ansolvate. Unlessotherwise stated, all temperatures are in degrees Celcius (° C.) and thefollowing abbreviations have the following definitions:

DSC Differential scanning calorimetryDVS Dynamic vapor sorptionHPLC High performance liquid chromatographyNA Not applicableND Not determinedQ Percent dissolved per unit timeRH Relative humidityRSD Residual standard deviationRRT Relative retention timeSS-NMR Solid state nuclear magnetic resonanceTGA Thermogravimetric analysisTG-IR Thermogravimetric infra red analysisXRPD X-ray powder diffractionVT-XRPD Variable temperature X-ray powder diffraction

Synthetic Routes for Preparing Compound 1

The compound of formula (I) was synthesized as schematically describedbelow and elaborated thereafter.

Example 1: Synthesis of Compound 15

To a solution of 2-bromobenzene-1,3-diol (5 g, 26.45 mmol) in DCM (50ml) at 0° C. was added DIPEA (11.54 mL, 66.13 mmol) and MOMCl (4.42 mL,58.19 mmol). The mixture was stirred at 0° C. for 1.5 h, and then warmedto room temperature. The solution was diluted with DCM, washed with sat.NaHCO₃, brine, dried and concentrated to give crude product, which waspurified by column (hexanes/EtOAc=4:1) to give desired product 15.58 g(90%).

Example 2: Synthesis of Compound 13 from 15

To a solution of 2-bromo-1,3-bis(methoxymethoxy)benzene (15) (19.9 g,71.8 mmol) in THF (150 mL) at −78° C. was added BuLi (2.5 M, 31.6 mL,79.0 mmol) dropwise. The solution was stirred at −78° C. for 25 min(resulting white cloudy mixture), then it was warmed to 0° C. andstirred for 25 min. The reaction mixture slowly turns homogenous. To thesolution was added DMF at 0° C. After 25 min, HPLC showed reactioncompleted. The mixture was quenched with sat. NH4Cl (150 mL), dilutedwith ether (300 mL). The organic layer was separated, aq layer wasfurther extracted with ether (2×200 mL), and organic layer was combined,washed with brine, dried and concentrated to give crude product, whichwas triturated to give 14.6 g desired product. The filtrate was thenconcentrated and purified by column to give additional 0.7 g, total massis 15.3 g.

Example 3: Synthesis of Compound 13 from Resorcinol 11

A three-necked round-bottom flask equipped with mechanical stirrer wascharged with 0.22 mol of NaH (50% suspension in mineral oil) undernitrogen atmosphere. NaH was washed with 2 portions (100 mL) of n-hexaneand then with 300 mL of dry diethyl ether; then 80 mL of anhydrous DMFwas added. Then 0.09 mol of resorcinol 11, dissolved in 100 mL ofdiethyl ether was added dropwise and the mixture was left under stirringat rt for 30 min. Then 0.18 mol of MOMCl was slowly added. After 1 hunder stirring at rt, 250 mL of water was added and the organic layerwas extracted with diethyl ether. The extracts were washed with brine,dried (Na₂SO₄), then concentrated to give the crude product that waspurified by silica gel chromatography to give compound 12 (93% yield).

A three-necked round-bottom flask was charged with 110 mL of n-hexane,0.79 mol of BuLi and 9.4 mL of tetramethylethylendiamine (TMEDA) undernitrogen atmosphere. The mixture was cooled at −10° C. and 0.079 mol ofbis-phenyl ether 12 was slowly added. The resulting mixture was leftunder magnetic stirring at −10° C. for 2 h. Then the temperature wasraised to 0° C. and 0.067 mol of DMF was added dropwise. After 1 h,aqueous HCl was added until the pH was acidic; the mixture was thenextracted with ethyl ether. The combined extracts were washed withbrine, dried (Na₂SO₄), and concentrated to give aldehyde 13 (84%).

2,6-bis(methoxymethoxy)benzaldehyde (13): mp 58-59° C. (n-hexane); IR(KBr) n: 1685 (C═O) cm⁻¹; ¹H-NMR (400 MHz, CDCl₃) δ 3.51 (s, 6H, 2OCH₃),5.28 (s, 4H, 2OCH₂O), 6.84 (d, 2H, J=8.40 Hz, H-3, H-5), 7.41 (t, 1H,J=8.40 Hz, H-4), 10.55 (s, 1H, CHO); MS, m/e (relative intensity) 226(M+, 3), 180 (4), 164 (14), 122 (2), 92 (2), 45 (100); Anal. Calc'd. forC₁₁H₁₄O₅: C, 58.40; H, 6.24. Found: C, 57.98; H, 6.20.

Example 4: The Synthesis of Compound 16

To a solution of 2,6-bis(methoxymethoxy)benzaldehyde (13) (15.3 g, 67.6mmol) in THF (105 mL) (solvent was purged with N₂) was added conc. HCl(12N, 7 mL) under N₂, then it was further stirred under N₂ for 1.5 h. Tothe solution was added brine (100 mL) and ether (150 ml). The organiclayer was separated and the aqueous layer was further extracted withether (2×200 mL). The organic layer was combined, washed with brine,dried and concentrated to give crude product, which was purified bycolumn (300 g, hexanes/EtOAc=85:15) to give desired product 16 (9.9 g)as yellow liquid.

Example 5: Synthesis of Compound 17

To a solution of 2-hydroxy-6-(methoxymethoxy)benzaldehyde (16) (10.88 g,59.72 mmol) in DMF (120 mL) (DMF solution was purged with N₂ for 10 min)was added K₂CO₃ (32.05 g, 231.92 mmol) and3-(chloromethyl)-2-(1-isopropyl-1H-pyrazol-5-yl)pyridine hydrochloride(10) (15.78 g, 57.98 mmol). The mixture was heated at 65° C. for 1.5 h,cooled to rt, poured into ice water (800 mL). The precipitated solidswere isolated by filtration, dried and concentrated to give desiredproduct (17, 18 g).

Example 6: Synthesis of Compound (I)

To a solution of2-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)-6-(methoxymethoxy)benzaldehyde(17) (18 g, 47.19 mmol) in THF (135 mL, solution was purged with N₂) wasadded conc. HCl (12N, 20 mL). The solution was stirred at rt for 3 hwhen HPLC showed the reaction complete. The mixture was added to asolution of NaHCO₃ (15 g) in water (1.2 L), and the resultingprecipitate was collected by filtration, dried to give crude solid,which was further purified by column (DCM/EtOAc=60:40) to give pureproduct (15.3 g).

Example 7: Synthesis of Compound I (Free Base) and its HCl Salt Form

Compound (I) free base (40 g) was obtained from the coupling of thealcohol intermediate 7 and 2,6-dihydroxybenzaldehyde 9 under Mitsunobuconditions. A procedure is also provided below:

Example 8: Synthesis of Compound (I) by Mitsunobu Coupling

Into a 2000-mL three neck round-bottom flask, which was purged andmaintained with an inert atmosphere of nitrogen, was placed a solutionof [2-[1-(propan-2-yl)-1H-pyrazol-5-yl]pyridin-3-yl]methanol (7) (70 g,322.18 mmol, 1.00 equiv) in tetrahydrofuran (1000 mL).2,6-Dihydroxybenzaldehyde (9) (49.2 g, 356.21 mmol, 1.10 equiv) and PPh₃(101 g, 385.07 mmol, 1.20 equiv) were added to the reaction mixture.This was followed by the addition of a solution of DIAD (78.1 g, 386.23mmol, 1.20 equiv) in tetrahydrofuran (200 ml) dropwise with stirring.The resulting solution was stirred overnight at room temperature. Theresulting solution was diluted with 500 ml of H₂O. The resultingsolution was extracted with 3×500 ml of dichloromethane and the combinedorganic layers were dried over sodium sulfate and concentrated undervacuum. The residue was applied onto a silica gel column with EA:PE(1:50-1:3) as eluent to yield the crude product. The crude product wasre-crystallized from i-propanol/H₂O in the ratio of 1/1.5. This resultedin 40 g (37%) of2-hydroxy-6-([2-[1-(propan-2-yl)-1H-pyrazol-5-yl]pyridin-3-yl]methoxy)benzaldehydeas a light yellow solid. The compound exhibited a melting point of80-82° C. MS (ES, m/z): 338.1 [M+1]. ¹H NMR (300 MHz, DMSO-d6) δ 11.72(s, 1H), 10.21 (s, 1H), 8.76 (d, J=3.6 Hz, 1H), 8.24 (d, J=2.7 Hz, 1H),7.55 (m, 3H), 6.55 (m, 3H), 5.21 (s, 2H), 4.65 (m, 1H), 1.37 (d, J=5.1Hz, 6H). ¹H NMR (400 MHz, CDCl₃) δ 11.96 (s, 1H), 10.40 (s, 1H), 8.77(dd, J=4.8, 1.5 Hz, 1H), 8.00 (d, J=7.8 Hz, 1H), 7.63 (d, J=1.8 Hz, 1H),7.49-7.34 (m, 2H), 6.59 (d, J=8.5 Hz, 1H), 6.37 (d, J=1.8 Hz, 1H), 6.29(d, J=8.2 Hz, 1H), 5.10 (s, 2H), 4.67 (sep, J=6.7 Hz, 1H), 1.50 (d,J=6.6 Hz, 6H).

In another approach, multiple batches of Compound (I) free base areprepared in multi gram quantities (20 g). The advantage of this route isthe use of mono-protected 2,6-dihydroxybenzaldehyde (16), whicheffectively eliminates the possibility of bis-alkylation side product.The mono-MOM ether of 2,6-dihydroxybenzaldehyde (16) can be obtainedfrom two starting points, bromoresorcinol (14) or resorcinol (11)[procedures described in the Journal of Organic Chemistry, 74(11),4311-4317; 2009]. All steps and procedures are provided below. Due tothe presence of phenolic aldehyde group, precautions (i.e., carry outall reactions under inert gas such as nitrogen) should be taken to avoidoxidation of the phenol and/or aldehyde group.

Preparation of compound I HCl salt: A solution of compound I (55.79 g,165.55 mmol) in acetonitrile (275 mL) was flushed with nitrogen for 10min, then to this solution was added 3N aqueous HCl (62 mL) at roomtemperature. The mixture was stirred for additional 10 min after theaddition, most of the acetonitrile (about 200 mL) was then removed byevaporation on a rotary evaporator at around 32° C., the remainingsolution was frozen by cooling in an acetone-dry ice bath andlyophilized to afford compound I HCl salt (59.4 g).

Example 9: Characterization of the HCl Salt of Compound 1

Technique Details Result XRPD indexed HCl salt of Compound 1 Microscope— pale yellow solids, thin blades/tablets, birefringent ¹H NMR DMSO-d6consistent with structure, <0.01 moles MEK XRPD — HCl salt of Compound 1DVS — 0.03% gain upon equilibration at 5% RH 0.10% gain from 5 to 95% RH0.09% loss from 95 to 5% RH post XRPD HCl I + Free Base Form I

Example 10: Physical Stability of the HCl Salt of Compound 1 Exposed toWater

Time (all times Condition are approximated) Observation XRPD Resultcontacted w/ — sheet formation after — water 5 min water slurry about 5min Floating yellow Free base (FB) solids convert to I (indexed) whitesolids upon isolation vacuum dried about 1 day Remain FB I water slurryabout 6 days white, thin blades, FB I + FB II birefringent (B)

Example 11: Physical Stability of the HCl Salt of Compound 1 withGrinding

Condition Time Observation XRPD Result grinding, dry 30 min offwhite/pale yellow HCl I grinding, wet 30 min off white/pale yellow pasteHCl I + FB I

Example 12: Physical Stability of the HCl Salt of Compound 1 Exposed toElevated Temperature and/or Vacuum

Condition Time Observation XRPD Result RT vacuum  6 days pale yellow,blades/plates, B HCl I + FB I 30° C.  6 hrs pale yellow, blades/tablets,B HCl I 12 hrs pale yellow, blades/tablets, B HCl I + FB I 24 hrs paleyellow, blades/tablets, B HCl I + FB I 40° C.  6 hrs pale yellow,blades/tablets, B HCl I + FB I 12 hrs pale yellow, blades/tablets, B HClI + FB I 24 hrs pale yellow, blades/tablets, B HCl I + FB I 40° C.  6hrs pale yellow, blades/tablets, B HCl I + FB I vacuum 12 hrs paleyellow, blades/tablets, B HCl I + FB I 24 hrs pale yellow,blades/tablets, B HCl I + FB I 60° C.  6 days pale yellow blades, B HClI + FB I 60° C.  6 days pale yellow, blades, B; HCl I + FB I + vacuumirregular residue other free base form 100 to 20 min pH paper abovesample HCl I + FB I + 125° C. indicate acidic volatiles other free baseform

Example 13: Generation of the Free Base of Compound 1 from theDisproportionation of the HCl Salt of Compound 1 in Water (the StartingMaterial is the HCl Salt of Compound 1)

Method Observation XRPD Result 1. contacted with water 1. pale yellow,FB I 2. sonicated wets poorly 3. filtered and rinsed with water 2. white4. dried under N₂ for 10 minutes 3. — 5. vacuum RT, overnight 4. — 5.— 1. contacted with water 1. — FB I + other 2. sonicated for 5 minutes2. pale yellow, free base form 3. slurried for 10 minutes turned white4. filtered, rinsed with water 3. — 5. dried under N₂ for 10 minutes 4.— 6. vacuum RT, overnight 5. white 7. stored in freezer 6. — 7. — 1.slurry in water, RT, 8 days; 1. thick white FB II seeded with FB IIslurry 2. filtered, rinsed with water 2. — 3. vacuum RT, overnight 3. —2. sub sample of slurry 2. — FB II (indexed) 3. rinsed with water 3. —

Example 14: Characterization Form I of the Free Base of Compound 1

Technique Details Result XRPD indexed Free Base Form I XRPD — Free BaseForm I TGA 25 to 0.2% weight loss up to 100° C. 350° C. DSC 25 toendothermic event with onset near 97° C. 350° C. Hot Stage 22.7° C.initial, fines, birefringent Microscopy 91.2° C. increase in particlesize and birefringence 94.2° C. increase in particle size andbirefringence 95.7° C. melt onset, larger particles from initial heating96.1° C. melt continuation 96.3° C. melt complete, no crystallizationupon melting 68.7° C. fresh preparation, larger magnification 91.1° C.increase in birefringence 94.8° C. melt onset, larger particles,birefringent 95.4° C. melt continuation 95.9° C. only few crystalsremain, cooled to 92.6° C. 92.6° C. held for 2 to 3 minutes crystalgrowth to larger blades, - began heating 96.3° C. complete melt ¹H NMRDMSO-d6 consistent with structure DVS — 0.02% loss upon equilibration at5% RH 0.22% gain from 5 to 95% RH 0.22% loss from 95 to 5% RH post XRPDFree Base Form I + other Free Base Material

Example 15: Characterization of Form II of the Free Base of Compound 1

Technique Details Result XRPD indexed Free Base Form II XRPD initialFree Base Form II after 7 days Free Base Form II TGA 25 to 350° C. 0.1%weight loss up to 100° C. DSC 25 to 350° C. endothermic event with onsetnear 97° C. ¹H NMR DMSO-d6 consistent with structure

Example 16: Characterization of Material N of the Free Base of Compound1

Technique Details Result XRPD — Free Base Material N TGA 25 to 350° C.0.2% weight loss up to 100° C. DSC 25 to 350° C. endothermic event withonset near 94° C. ¹H NMR DMSO-d6 consistent with structure, no residualreaction solvent observed

Example 17: Competitive Interconversion Slurries Between Free Base FormsI and II

Conditions Solvent Observation XRPD Result 6° C., 6 days water white FBII 6° C., 6 days heptane white FB II 6° C., 6 days IPE faint pale FB Nyellow RT, 6 days water white FB II RT, 6 days heptane off white FB IIRT, 6 days IPE pale yellow FB N ^(Error! Bookmark not defined.) RT, 6days toluene pale yellow FB N 57° C., 2 days water fines, off FB II + FBI white, B 57° C., heptane blades and FB II overnight tablets, B 57° C.,IPE blades, FB II overnight laminated, pale yellow, B

Example 18: Competitive Interconversion Slurries Between Free Base FormII and Material N

Conditions 35° C., 3 days heptane pale yellow fines, B FB N 57° C., 3days heptane larger blades, and rosettes of blades, B FB II

Example 19: Selected Experimental Methods

Indexing:

XRPD patterns are indexed by using proprietary SSCI software. Agreementbetween the allowed peak positions, marked with red bars within thefigures, and the observed peaks indicates a consistent unit celldetermination. Indexing and structure refinement are computationalstudies which are performed under the “Procedures for SSCI Non-cGMPActivities.” To confirm the tentative indexing solution, the molecularpacking motifs within the crystallographic unit cells must bedetermined. No attempts at molecular packing were performed.

Differential Scanning Calorimetry (DSC):

DSC was performed using a TA Instruments Q2000 differential scanningcalorimeter. Temperature calibration was performed using NIST-traceableindium metal. The sample was placed into an aluminum DSC pan, coveredwith a lid, and the weight was accurately recorded. A weighed aluminumpan configured as the sample pan was placed on the reference side of thecell. The data acquisition parameters and pan configuration for eachthermogram are displayed in the image in the Data section of thisreport. The method code on the thermogram is an abbreviation for thestart and end temperature as well as the heating rate; e.g., −30-250-10means “from −30° C. to 250° C., at 10° C./min”. The following summarizesthe abbreviations used in each image for pan configurations: Tzerocrimped pan (TOC); and Lid not crimped (NC).

Dynamic Vapor Sorption (DVS):

Dynamic vapor sorption (DVS) data were collected on a VTI SGA-100 VaporSorption Analyzer. NaCl and PVP were used as calibration standards.Samples were not dried prior to analysis. Adsorption and desorption datawere collected over a range from 5 to 95% RH at 10% RH increments undera nitrogen purge. The equilibrium criterion used for analysis was lessthan 0.0100% weight change in 5 minutes with a maximum equilibrationtime of 3 hours. Data were not corrected for the initial moisturecontent of the samples.

Microscopy

Hot Stage Microscopy:

Hot stage microscopy was performed using a Linkam hot stage (FTIR 600)mounted on a Leica DM LP microscope equipped with a SPOT Insight™ colordigital camera. Temperature calibrations were performed using USPmelting point standards. Samples were placed on a cover glass, and asecond cover glass was placed on top of the sample. As the stage washeated, each sample was visually observed using a 20×0.40 N.A. longworking distance objective with crossed polarizers and a first order redcompensator. Images were captured using SPOT software (v. 4.5.9).

Polarized Light Microscopy:

During the course of experimentation generated samples were viewedutilizing a microscope with cross polarized light to observe morphologyand birefringence. Samples were visually observed at 40× magnification.

¹H Solution Nuclear Magnetic Resonance (¹H NMR)

SSCI:

Samples were prepared for NMR spectroscopy as ˜5-50 mg solutions in theappropriate deuterated solvent. The specific acquisition parameters arelisted on the plot of the first full spectrum of each sample in the datasection for samples run at SSCI.

Spectral Data Solutions:

For samples run using Spectral Data Solutions (subcontractor), thesolution ¹H NMR spectra were acquired at ambient temperature on a Varian^(UNITY)INOVA-400 spectrometer (¹H Larmor Frequency=399.8 MHz). Thespecific acquisition parameters are listed on the spectral data sheetand on each data plot of the spectrum of the sample.

Thermogravimetric Analysis (TGA)

TG analyses were performed using a TA Instruments 2950 thermogravimetricanalyzer. Temperature calibration was performed using nickel andAlumel™. Each sample was placed in an aluminum pan and inserted into theTG furnace. The furnace was heated under a nitrogen purge. The dataacquisition parameters are displayed above each thermogram in the Datasection of this report. The method code on the thermogram is anabbreviation for the start and end temperature as well as the heatingrate; e.g., 25-350-10 means “from 25° C. to 350° C., at 10° C./min”. Theuse of 0 as the initial temperature indicates sample run initiated fromambient.

XRPD Analysis

INEL:

XRPD patterns were collected with an Inel XRG-3000 diffractometer. Anincident beam of Cu Kα radiation was produced using a fine-focus tubeand a parabolically graded multilayer mirror. Prior to the analysis, asilicon standard (NIST SRM 640d) was analyzed to verify the Si 111 peakposition. A specimen of the sample was packed into a thin-walled glasscapillary, and a beam-stop was used to minimize the background from air.Diffraction patterns were collected in transmission geometry usingWindif v. 6.6 software and a curved position-sensitive Equinox detectorwith a 20 range of 120°. The data-acquisition parameters for eachpattern are displayed above the image in the Data section of thisreport.

PANalytical Transmission:

XRPD patterns were collected with a PANalytical X'Pert PRO MPDdiffractometer using an incident beam of Cu radiation produced using anOptix long, fine-focus source. An elliptically graded multilayer mirrorwas used to focus Cu Kα X-rays through the specimen and onto thedetector. Prior to the analysis, a silicon specimen (NIST SRM 640d) wasanalyzed to verify the Si 111 peak position. A specimen of the samplewas sandwiched between 3 m thick films and analyzed in transmissiongeometry. A beam-stop, short antiscatter extension, and an antiscatterknife edge were used to minimize the background generated by air. Sollerslits for the incident and diffracted beams were used to minimizebroadening from axial divergence. Diffraction patterns were collectedusing a scanning position-sensitive detector (X'Celerator) located 240mm from the specimen and Data Collector software v. 2.2b. Thedata-acquisition parameters for each pattern are displayed above theimage in the Data section of this report including the divergence slit(DS) before the mirror and the incident-beam antiscatter slit (SS).

PANalytical Reflection:

XRPD patterns were collected with a PANalytical X′Pert PRO MPDdiffractometer using an incident beam of Cu Kα radiation produced usinga long, fine-focus source and a nickel filter. The diffractometer wasconfigured using the symmetric Bragg-Brentano geometry. Prior to theanalysis, a silicon specimen (NIST SRM 640d) was analyzed to verify theobserved position of the Si 111 peak is consistent with theNIST-certified position. A specimen of the sample was prepared as athin, circular layer centered on a silicon zero-background substrate.Antiscatter slits (SS) were used to minimize the background generated byair. Soller slits for the incident and diffracted beams were used tominimize broadening from axial divergence. Diffraction patterns werecollected using a scanning position-sensitive detector (X'Celerator)located 240 mm from the sample and Data Collector software v. 2.2b. Thedata acquisition parameters for each pattern are displayed above theimage in the Data section of this report including the divergence slit(DS) and the incident-beam SS.

Approximate Solubility:

A weighed sample was treated with aliquots of the test solvent at roomtemperature. The mixture was sonicated between additions to facilitatedissolution. Complete dissolution of the test material was determined byvisual inspection. Solubility was estimated based on the total solventused to provide complete dissolution. Some samples were then heated andobserved visually for complete dissolution. The actual solubility may begreater than the value calculated because of the use of solvent aliquotsthat were too large or due to a slow rate of dissolution. The solubilityis expressed as “less than” if dissolution did not occur during theexperiment. If complete dissolution was achieved as a result of only onealiquot addition, the solubility is expressed as “greater than”.

Anti-Solvent Additions:

Compound 1/organic solvent solutions were contacted with solvents thatCompound 1 was determined to be poorly soluble or insoluble in. Theseanti solvent additions were added to help lower the solubility of thesolvent system and induce crystallization.

Cooling and Slow Cools:

Solutions were prepared in the selected solvent or solvent/anti-solventsystem. These solutions were chilled below room temperature within arefrigerator for varying lengths of time in an attempt to inducenucleation. The presence or absence of solids was noted. Uponobservation of solids, in quantities sufficient for analysis, isolationof material was conduction. If insufficient quantities were presentfurther cooling was performed in a freezer. Samples were either isolatedfor analysis wet or as dry powders.

Compression:

Selected samples were compressed utilizing a KBr die and a Carverhydraulic press. An applied load of 10000 lbs was applied to the dieshaft for approximately 20 minutes.

Crystallization from Solution:

Saturated solutions were generated at ambient and then capped.Nucleation was observed to occur from these systems during evaluation ofthe Free Base of Compound 1.

Fast Evaporation:

Solutions were prepared in selected solvents and agitated betweenaliquot additions to assist in dissolution. Once a mixture reachedcomplete dissolution, as judged by visual observation, the solution wasallowed to evaporate at ambient temperature in an uncapped vial or atambient under nitrogen. The solids that formed were isolated forevaluation.

Milling:

Selected material was milled utilizing a Reitch Mill. The material wasloaded into an agate lined milling vessel followed by the addition of anagate ball. The vessel was then placed on to the mill and milled forapproximately 30 minutes at frequency of 1/30 seconds. The milling wasstopped approximately every 10 minutes and material scraped from thewall before further milling.

Slurry:

Solutions were prepared by adding enough solids to a given solvent sothat excess solids were present. The mixture was then agitated in asealed vial at either ambient or an elevated temperature. After a givenamount of time, the solids were isolated for analysis.

Temperature and Relative Humidity Stress:

Selected materials were stressed at elevated related humidity and/ortemperature. Relative humidity jars (saturated salt solutions used togenerate desired relative humidity) were utilized to store selectedsamples. The following relative humidity jars were utilized duringevaluation: 75% RH (NaCl) and 60% (NaBr), to investigate the effects ofhumidity. Temperatures utilized were ambient, 30, 40, 60, and 100-125°C.

Vacuum:

Selected materials were stressed under reduced pressure for a set timeperiod. Initial stressing was conducted with the in-house vacuum systemwith absolute pressure readings <500 mTorr, typically 30 to 50 mTorr(0.030 to 0.05 mm Hg). Additional vacuum stressing was conducted at 48mmHg utilizing a portable lab vacuum and bleed to simulate conditionssimilar to those expected during process.

Example 20: Disproportionation of the HCl Salt

The disproportionation of the HCl salt in water was utilized to generatefree base. The nucleation of Free Base Form I occurs first. Extendingthe slurry time induces the transformation to a more thermodynamicallystable phase relative to Form I, Free Base Form II.

Three anhydrous materials of the free base were identified; Free BaseForms I, II, and Material N. Free Base Material N appears to be moststable form, relative to Forms I and II, at room temperature. Free BaseMaterial N is enantiotropic relative to Form II, and will transformreversibly at a specific transition temperature (estimated near 42° C.).Above the transition temperature, Free Base Form II appears to be themost stable form, relative to Form I and Material N.

The HCl salt (termed “HCl Form I”) was subjected to various stressconditions and monitored by XRPD to evaluate physical stability. Asdiscussed, disproportionation occurred during the DVS experiment of theHCl salt, indicating instability upon exposure to elevated humidity.Disproportionation is further evident with wet milling or in directcontact with water (e.g. slurry) as shown by the presence of Free BaseForms I or II, identified by XRPD. The volatilization and loss of HClupon heating and/or vacuum is shown by the presence of Free Base Form I,identified by XRPD, and also indicates instability at these conditions.

-   -   Contact with water resulted in a visual color change of the        material from pale yellow to white; physical changes were also        observed microscopically. Immediate disproportionation occurs.        XRPD analysis identified the resulting material from a water        slurry (˜5 minutes) as Free Base Form I. Free Base Form II also        becomes evident if the amount of time in the slurry is extended.    -   The volatilization of HCl was evident within hours of exposure        to drying conditions. Conversion to Free Base Form I was        observed by XRPD at 30° C. (after 12 hrs), 40° C. (after 6 hrs),        and at 40° C./48 mmHg (after 6 hrs).    -   Free Base Material C becomes evident at more extreme conditions        involving elevated temperatures. Heating HCl Form I up to        125° C. induces the loss of acidic volatiles (judged visually by        use of pH paper held above sample). XRPD analysis identifies the        resulting sample as a mixture of HCl Form I, Free Base Form I,        and Free Base Material C. Exposing the HCl salt to 60° C. under        vacuum for 6 days provides the same result. The nature of        Material C is not established

The HCl salt was shown to disproportionate immediately in water. Thisphenomenon was utilized to generate free base. The nucleation of FreeBase Form I occurs first. Extending the slurry time induces thetransformation to a more thermodynamically stable phase relative to FormI, Free Base Form II.

-   -   A 20 ml vial was charged with 266.4 mg of HCl Form I and        contacted with 10 ml of water. The sample was sonicated until        the pale yellow material changed color to white. The resulting        solids were collected by filtration (water aspirated) and rinsed        with 10 ml of water. A nitrogen purge was blown over the sample        for approximately 10 minutes prior to exposure to vacuum at        ambient temperature to dry overnight. The resulting material was        analyzed by XRPD and determined to be Free Base Form I.    -   A 250 ml Erlenmeyer flask was charged with 6.0250 grams of HCl        Form I and contacted with 220 mL of water. The sample was        sonicated for approximately 5 minutes to disperse the material.        The yellow material changed color to white during sonication. A        stir bar was added and the sample was stirred at 700 RPM for        approximately 10 minutes. The solids were collected by        filtration and rinsed with 220 ml of water followed by a        nitrogen purge over the sample for approximately 10 minutes        prior to exposure to vacuum at ambient temperature. The sample        was dried at this condition for approximately 24 hours yielding        5.1834 grams of material. The resulting material was analyzed by        XRPD and determined to be a mixture of Free Base Form I and Free        Base Material D. (The nature of Material D is not established.)

The procedure used to generate Free Base Form II is described below.

-   -   A 20 ml vial was charged with 477.5 mg of HCl Form I lot 20 and        contacted with 20 ml of water. The sample was sonicated until        the pale yellow material changed color to white. A small amount        of sample (mixture of Free Base Forms I and II) was added as        seeds. A stir bar was added and the sample was stirred at 200        RPM for 8 days. The resulting solids were collected by        filtration (water aspirated) and rinsed with 15 ml of water. The        sample was exposed to vacuum at ambient temperature to dry        overnight. The resulting material was analyzed by XRPD and        determined to be Free Base Form II.

Example 21: Additional Procedures for the Preparation of the Free Baseof Form I, Form II, and From N Conversion of the Free Base of Compound 1to the HCl Salt

General Procedure:

Slowly treat a solution of the free base of Compound 1 in MEK (5 vol)with conc HCl (1.5 eq). Cool the resulting slurry to 0-5° C. for 1 h andfilter. Wash solids with MEK (1 vol). Dry under vacuum at 30-35° C.

Preparation A:

Following the general procedure above, 35 g of crude Compound 1 wasprocessed to provide the HCl salt as a pale yellow solid (32.4 g, 82%yield, 99.8% purity by HPLC).

Preparation of the Free Base Form I from the HCl Salt of Compound 1

General Procedure:

Vigorously stir a slurry of the HCl salt of Compound 1 in DIW (10 vol)for 5 min to 2 h. Filter the slurry, wash with DIW (2×1 vol), dry onfunnel, then further dry under vacuum at 30-35° C.

Preparation A:

Following the general procedure above, after stirring for 1 h, 32 g ofthe HCl salt of Compound 1 was processed to provide the free base as apale yellow solid (27.3 g, 95% yield, 99.8% purity by HPLC; DSCindicates Form I).

Preparation B:

Following the general procedure above, after stirring for 1 h, 39 g ofthe HCl salt of Compound 1 was processed to provide the free base as apale yellow solid (31.8 g, 90% yield, >99.9% purity by HPLC)).

Preparation C:

Thus, the HCl salt of Compound 1 (134 g) was vigorously stirred in water(10 vol) until the material appeared as a finely dispersed white slurry.After filtration and drying, a white crystalline solid (116 g, 96%recovery, >99.9% purity by HPLC) was isolated.

Preparation D:

The purpose of this experiment was to prepare the free base fromCompound 1, HCl. Thus, the HCl salt of Compound 1 (65.3 g) wasvigorously stirred in water (10 vol) until the material appeared as afinely dispersed white slurry. After filtration and drying, a whitecrystalline solid (57.5 g, 97.6% recovery, >99.9% purity by HPLC) wasisolated.

Preparation of GBT000440 Free Base Form II from GBT000440 Free Base FormI

General Procedure:

Stir a slurry of the free base of Compound 1 Form I in an appreciatesolvent (e.g. heptane or water) (10 vol) for 1-7 days. Filter theslurry, wash with DIW (2×1 vol), dry on funnel, then further dry undervacuum at 30-35° C.

Preparation A:

Thus, the free base of Compound 1, Form I (114 g) was stirred inn-heptane (10 vol) at 35° C. After 4 days, XRPD indicated the materialwas Form II. The slurry was filtered and dried to provide 110 g offwhite solid.

Preparation B:

the free base of Compound 1 (5 g) was slurried in heptanes (10 vol 50mlL) at room temperature. After 4 days, the slurry was filtered toprovide an off-white solid.

Preparation C:

the free base of Compound 1 (5.8 kg) was slurried in heptanes (10 vol)at room temperature. After 2 days, the slurry was filtered and washedwith 2×2 vol n-heptane to provide 4.745 kg of Form II as an off-whitesolid.

Preparation D:

the free base of Compound 1 (5 g) was slurried in water. After 4 days,the slurry was filtered to provide an off-white solid.

Preparation of GBT000440 Free Base Form N from GBT000440 Free Base FormI or Form II

General Procedure:

Stir a slurry of the free base of Compound 1, Form I in MTBE (4 vol) atroom temperature for at least 4 days. After 4 days, filter the slurry toprovide an off-white solid. Obtain XRPD to confirm polymorph as MaterialN.

Preparation A:

Following the general procedure above, 27 g of the free base of Compound1, Form I (48TRS079) was stirred in MTBE at 18-23° C. for 4 days. DSCindicated it should be Material N. Isolated 22.2 g cream colored solid(82% recovery, 99.9 purity by HPLC). XRPD analysis planned.

Preparation B:

Following the general procedure above, 31 g of the free base of Compound1, Form I was stirred in 3 vol MTBE at 18-23° C. for 4 days.

Preparation C:

the free base of Compound 1, Form I (13KWB023, 1 g) was slurried in MTBE(5 vol) at room temperature. Slurry was seeded with Material N (50 mg).After 4 days, the slurry was filtered to provide a off-white solid. DSCindicated the melting point was the same as Material N.

Preparation D:

The purpose of this experiment was to convert the free base of Compound1, Form II to Material N. Thus, the free base of Compound 1 (0.5 g) wasstirred in 5 vol of di-n-propyl ether at 18-23° C. After 2 days, DSCcorresponded to the pattern observed for Material N. XRPD analysisconfirmed Material N had been formed.

Preparation E:

To the free base of Compound 1, Form II (5 g) was added diisopropylether (5 vol, 25 mL) at room temperature. After 4 days, the slurry wasfiltered to provide a off-white solid. DSC indicates Material N.

Example 22: Preliminary Solvent-Based Screens

Rapid, solvent-based screens were conducted in an attempt to determinethe most stable form of the free base of Compound 1. The study alsoprovides a preliminary assessment of the propensity of these materialsto exist in various crystal forms. Generated solids were observed bypolarized light microscopy (PLM) and/or analyzed by X-ray powderdiffraction (XRPD), comparing the resulting XRPD patterns to knownpatterns of Compound 1.

If possible, XRPD patterns were indexed. Indexing is the process ofdetermining the size and shape of the crystallographic unit cell giventhe peak positions in a diffraction pattern. The term gets its name fromthe assignment of Miller index labels to individual peaks. XRPD indexingserves several purposes. If all of the peaks in a pattern are indexed bya single unit cell, this is strong evidence that the sample contains asingle crystalline phase. Given the indexing solution, the unit cellvolume may be calculated directly and can be useful to determine theirsolvation states. Indexing is also a robust description of a crystallineform and provides a concise summary of all available peak positions forthat phase at a particular thermodynamic state point.

Materials exhibiting unique crystalline XRPD patterns, based on visualinspection of peaks associated with these materials, were given letterdesignations. The letter designation is tentatively associated with theword ‘Material’ if insufficient characterization data is available. Thenomenclature is used only to aid in the identification of unique XRPDpatterns and does not imply that the stoichiometry, crystalline phasepurity, or chemical purity of the material is known. Materials arefurther designated as forms with Roman numeral designations (i.e., FreeBase Material A=Free Base Form I), when phase purity (obtained throughindexing of the XRPD pattern or single crystal structure elucidation)and chemical identity/purity (obtained through proton NMR spectroscopy)of the material is determined.

Three anhydrous materials were identified: Forms I, II, and Material N.Material N appears to be most stable form, relative to Forms I and II,at room temperature. Material N is enantiotropic relative to Form II,and will transform reversibly at a specific transition temperature(estimated near 42° C.). Above the transition temperature, Form IIappears to be the most stable form, relative to Form I and Material N.

Materials C and D are used to identify a few additional, low intensitypeaks observed in XRPD patterns which were predominantly composed of theFree Base Form I of Compound 1 or mixtures of the HCl Form I and FreeBase Form I of Compound 1.

Example 23: Anhydrous Ansolvate Forms Form I

Free Base Form I is a metastable, anhydrous phase of the free base thatis formed immediately from the disproportionation of the HCl salt inwater. A representative XRPD pattern of Form I was successfully indexedand the unit cell volume is consistent with anhydrous free base. Visualcomparison of the XRPD pattern to the historical pattern of the freebase provided indicates the material may be similar; however, thehistorical pattern appears to exhibit additional peaks from a potentialmixture.

The ¹H NMR spectrum is consistent with the chemical structure ofCompound 1. The chemical shift at approximately 2.5 ppm is assigned toDMSO (due to residual protons in the NMR solvent). Peaks that could beassociated with residual solvents were not visible, consistent with theanhydrous unit cell volume determined from the indexing solution aboveand the negligible wt % loss observed by TGA discussed below.

Thermograms (TG) data shows negligible weight loss, 0.2%, up to 100° C.,consistent with an anhydrous form. The DSC exhibits a single endothermwith an onset near 97° C. (similar to what is observed for Form II). Theendotherm is consistent with a melt by hot stage microscopy. However,changes in particle size and birefringence were evident prior to themelt; a possible phase change occurred. Consequently, if a phase changeoccurred and an endotherm similar to that of Free Base Form II wasobserved, it can be inferred that the observed melt is truly not of FormI but of the resulting phase, most likely Form II.

The DVS isotherm indicates Form I is not hygroscopic. Negligible weightgain and loss, 0.2%, was observed through sorption/desorption. By XRPD,the material recovered from the DVS experiment was predominately FreeBase Form I with a few additional peaks. The additional peaks weretermed Free Base Material D. The nature of Material D is unknown;however, the appearance of another phase(s) indicates that Form I is notlikely physically stable at elevated humidity conditions (at ambienttemperature).

Form II

Free Base Form II is an anhydrous phase of the free base. Form II isenantiotropically related to Material N, where it is thethermodynamically stable form above an estimated transition temperatureof 42° C. Form II can be generated in solvents that do not form knownsolvates; such as heptane, IPE, MTBE, or toluene; through short-termslurry conversions of Form I (where the crystallization kinetics delaythe nucleation of the more stable form) or elevated temperature slurries(above 42° C.). A representative XRPD pattern of Form II wassuccessfully indexed and the unit cell volume is consistent withanhydrous free base of Compound 1.

The ¹H NMR spectrum is consistent with the chemical structure ofCompound 1. The chemical shift at approximately 2.5 ppm is assigned toDMSO (due to residual protons in the NMR solvent). Peaks that could beassociated with residual solvents were not visible, consistent with theanhydrous unit cell volume determined from the indexing solution aboveand the negligible wt % loss observed by TGA discussed below.

Thermograms (TG) data show negligible weight loss, 0.1%, up to 100° C.,consistent with an anhydrous form. The DSC exhibits a single endotherm(80.1 J/g) with an onset near 97° C.

Form II remained unchanged after 7 days at ambient storage, throughreanalysis by XRPD. The form is known to be thermodynamicallymetastable, relative to Material N, at this condition; however, thekinetics of polymorph conversion may be slow at ambient conditions inthe solid state.

Material N

Free Base Material N is an anhydrous phase of the free base. Material Nis enantiotropically related to Form II, where it is thethermodynamically stable form below an estimated transition temperatureof 42° C. Given the opportunity, Material N can be generated throughslurries in solvents that do not form known solvates; such as heptane,IPE, MTBE, or toluene; at temperatures below 42° C. The following is anexample of a laboratory scale procedure used to generate Free BaseMaterial N.

-   -   53.0 mg of Free Base Form I was contacted with 2 ml of an        IPE/free base solution (concentration 13 mg/ml). A stir bar was        added and the sample was slurried for 7 days at ambient. The        solution was decanted from the sample and the remaining solids        briefly dried under nitrogen. Characterization Data indicates        Material N is a unique crystalline phase.

The ¹H NMR spectrum is consistent with the chemical structure ofCompound 1. The chemical shift at approximately 2.5 ppm is assigned toDMSO (due to residual protons in the NMR solvent). Peaks that could beassociated with residual solvents were not visible, consistent with thenegligible wt % loss observed by TGA discussed below.

Thermograms (TG) data show negligible weight loss, 0.2%, up to 100° C.,consistent with an anhydrous form. The DSC exhibits a single endotherm(82.8 J/g) with an onset at 94° C.

Tentative Determination of the Thermodynamic Relationship between FreeBase Forms I, II, and Material N

Characterization data indicates that Forms I, II, and Material N areunique crystalline phases; however, only the XRPD patterns of Forms Iand II were successfully indexed to confirm phase purity. Therefore, anyproposed thermodynamic relationship between these materials is a workinghypothesis, where the phase purity of Material N is assumed.

Phase transitions of solids can be thermodynamically reversible orirreversible. Crystalline forms which transform reversibly at a specifictransition temperature (T_(p)) are called enantiotropic polymorphs. Ifthe crystalline forms are not interconvertable under these conditions,the system is monotropic (one thermodynamically stable form). Severalrules have been developed to predict the relative thermodynamicstability of polymorphs and whether the relationship between thepolymorphs is enantiotropic or monotropic. The heat of fusion rule isapplied within this study. The heat of fusion rule states that if thehigher melting form has the lower heat of fusion then the two forms areenantiotropic, otherwise they are monotropic.

Material N appears to be most stable form, relative to Forms I and II,at room temperature. Based on the heats of fusion and melts determinedby DSC, Material N is enantiotropic relative to Form II, and willtransform reversibly at a specific transition temperature (T^(N-II)).Due to a possible phase change of Form I to Form II that occurred priorto the observed endotherm in the DSC, the relationship of Form I witheither Material N or Form II cannot be conclusively determined throughthe heat of fusion rule. However, through various interconversionslurries, it was shown that Form I is the least thermodynamically stableform between 6° C. and T^(N-II). In addition, assuming that Form Ispontaneously converted to Form II in the DSC at elevated temperatures(prior to the observed melt), it must follow that Form II is also morestable than Form I above T^(N-II).

Example 24: Estimated Transition Temperature

The estimated transition temperature between two enantiotropicallyrelated forms can be calculated from their melt onsets and heats offusion based on the equation shown below.

$T_{p} = \frac{{\Delta \; H_{f,2}} - {\Delta \; H_{f,1}} + {\left( {C_{p,{liq}} - C_{p,1}} \right) \cdot \left( {T_{f,1} - T_{f,2}} \right)}}{\frac{\Delta \; H_{f,2}}{T_{f,2}} - \frac{\Delta \; H_{f,1}}{T_{f,1}} + {\left( {C_{p,{liq}} - C_{p,1}} \right) \cdot {\ln \left( \frac{T_{f,1}}{T_{f,2}} \right)}}}$Where, (C_(p, liq) − C_(p, 1)) = k ⋅ Δ H_(f, 1) and  k = 0.005

Between Material N and Form II, the equation estimates a transitiontemperature of 42° C. To summarize, the relative stability of the formsfrom most to least stable is shown below.

Temperature Relative Range* Stability Comments Below 6° C. N > IIRelationships to Form I are not established below this temp Between 6°C. and N > II > I — T^(N-II) Above T^(N-II) (II > N) and Relationshipbetween Form (II > I) I and Material N is not established above thistemp *T^(N-II) is estimated to be near 42° C.

Example 25: Energy—Temperature Diagram

The Energy—Temperature Diagram of FIG. 17 is a semi-quantitativegraphical solution of the Gibbs-Helmholtz equation, where the enthalpy(H) and free energy (G) isobars for each form are depicted as a functionof temperature.

Example 26: Competitive Interconversion Slurry Experiments

Interconversion experiments were performed to support the thermodynamicrelationship between polymorphs illustrated by the Energy—TemperatureDiagram above. Interconversion or competitive slurry experiments are asolution-mediated process that provides a pathway for the less soluble(more stable) crystal to grow at the expense of the more soluble crystalform. Outside the formation of a solvate or degradation, the resultingmore stable polymorph from an interconversion experiment is contemplatedto be independent of the solvent used because the more thermodynamicallystable polymorph has a lower energy and therefore lower solubility. Thechoice of solvent affects the kinetics of polymorph conversion and notthe thermodynamic relationship between polymorphic forms.

The results of the interconversion studies are consistent with thetentative Energy—Temperature Diagram shown herein. Binary slurries wereprepared at ambient, 6, and 57° C. using Forms I and II. Form IIresulted from the majority of these experiments, confirming that Form IIis more stable relative to Form I within this temperature range.

A few of the experiments conducted at ambient and 6° C. resulted inMaterial N. Although this does not provide specific clarificationbetween Forms I and II, it does provide evidence that Material N is themost stable form relative to both Forms I and II at these temperatures(which were conducted below the estimated transition temperature of 42°C.). Additional interconversion slurries between Form II and Material Nwere conducted at temperatures which bracket this estimated transitiontemperature and confirm that Form II and Material N areenantiotropically related.

Example 27: Solid-state Nuclear Magnetic Resonance

¹³C and ¹⁵N spectra acquired for the three polymorphic forms I, II andMaterial N. See FIGS. 10 and 11. Spectra were acquired at 253K toprevent any low temperature transitions occurring during measurement andacquisition parameters optimised for each polymorphic form.

Based on solid-state nuclear magnetic resonance, all three forms arecrystalline and are distinct polymorphic forms. Form I contains onemolecule per asymmetric unit, Form II contains two molecules perasymmetric unit and Form N contains four molecules per asymmetric unit.See the ¹⁵N spectra in FIG. 11.

Example 28: Chemical and Physical Stability Evaluation of the Free BaseForm I of Compound 1

A mixture predominately composed of Free Base Form I (with Free BaseMaterial D) were exposed to stability conditions to assess physical andchemical stability. Three conditions were used; open to 25° C./60% RH,open to 40° C./75% RH, and closed to 60° C. Physical stability wasevaluated by XRPD. Chemical stability was determined through UPLC and ¹HNMR, when applicable. Materials were tested after 1, 7, and 14 days ofexposure.

Chemical Stability of Free Base Form I

For the free base stability sample, UPLC showed very low levels ofimpurities present. The level of impurities did not rise significantlyafter 14 days of age. This would seem to indicate good chemicalstability against the conditions used for stability assessment. The ¹HNMR spectra of samples exposed to 60° C. (14 days) were also consistentwith this conclusion.

Physical Stability of Free Base Form I

The free base of Compound 1 remained unchanged, by XRPD, at 25° C./60%RH. However, differences were observed at the other two conditions. Thefew, minor peaks attributed to Free Base Material D were no longerobserved, indicating that Material D is metastable and is not sustainedat elevated temperatures. In addition, Free Base Form II was observedafter 7 days of age. This is consistent with the conclusions discussedherein, where Free Base Form II is more stable relative to Free BaseForm I at these temperatures.

Example 29: Physical Stability Evaluation of the Free Base Form II andMaterial N (Form N) of Compound 1

DSC was modulated at low underlying heating rate, followed by X-raypowder diffraction. A low underlying heating rate was used of 0.02° C.min⁻¹. The temperature was 80° C. for form N and 90° C. for form II.Exposure was essentially isothermal, covering a temperature range withsensitivity to detect changes in physical form. The resultant materialswere examined by X-ray powder diffraction. No changes in physical formwere observed for either polymorphic form II or polymorphic form N(i.e., material N).

Forms II and N were exposed to 40° C./75% relative humidity (RH), 80°C., 80° C./80% RH for 9 days followed by X-ray powder diffraction. Nochanges in physical form were observed for either polymorphic form II orpolymorphic form N.

The thermodynamic barrier for inter-conversion between polymorphic formII and form N is high, and physical stability is good for both forms.Thermally induced inter-conversion between form II and form N isunlikely to occur.

Example 30: The Relative Thermodynamic Stability of Polymorphic Forms IIand N

Extended solvent mediated maturation studies were conducted with 1:1 w/wmixtures of polymorphic form II and form N. Hexane provided a goodmedium for solvent assessments. The temperatures used include −20° C.,−10° C., 0° C., 10° C., 20° C., 30° C., 40° C. and 50° C. Significantlyincreased solubility was observed at 30° C., 40° C. and 50° C. Solidsderived from maturation at −20° C., −10° C., 0° C., 10° C., 20° C. wereanalyzed by X-ray powder diffraction. In each case, significantconversion to Form N was observed.

Form N is thermodynamically more stable than form II at temperatures of20° C. and lower. An enantiotropic relationship between the two forms islikely to exhibit equivalence in thermodynamic stability at ca. 30-40°C.

Example 31: Morphology of Form N

Initial examination of a batch of polymorphic form N indicates anacicular morphology.

While this invention has been described in conjunction with specificembodiments and examples, it will be apparent to a person of ordinaryskill in the art, having regard to that skill and this disclosure, thatequivalents of the specifically disclosed materials and methods willalso be applicable to this invention; and such equivalents are intendedto be included within the following claims.

1.-20. (canceled)
 21. A crystalline ansolvate of Compound 1:

characterized by at least two X-ray powder diffraction peaks (Cu Kαradiation) selected from 11.65°, 11.85°, 12.08°, 16.70°, 19.65° and23.48 °2θ (each ±0.2 °2θ).
 22. The crystalline ansolvate of claim 21,characterized by at least three X-ray powder diffraction peaks (Cu Kαradiation) selected from 11.65°, 11.85°, 12.08°, 16.70°, 19.65° and23.48 °2θ (each ±0.2 °2θ).
 23. The crystalline ansolvate of claim 21,characterized by an X-ray powder diffraction pattern (Cu Kα radiation)substantially similar to FIG.
 7. 24. The crystalline ansolvate of claim21, characterized by an endothermic peak at 95° C. ±2° C. as measured bydifferential scanning calorimetry.
 25. A pharmaceutical compositioncomprising a pharmaceutically acceptable excipient and a crystallineansolvate of Compound 1:

characterized by at least two X-ray powder diffraction peaks (Cu Kαradiation) selected from 11.65°, 11.85°, 12.08°, 16.70°, 19.65° and23.48 °2θ (each ±0.2 °2θ).
 26. The pharmaceutical composition of claim25, wherein the crystalline ansolvate of Compound 1 is characterized byat least three X-ray powder diffraction peaks (Cu Kα radiation) selectedfrom 11.65°, 11.85°, 12.08°, 16.70°, 19.65° and 23.48 °2θ (each ±0.2°2θ).
 27. The pharmaceutical composition of claim 25, wherein thecrystalline ansolvate of Compound 1 is characterized by an X-ray powderdiffraction pattern (Cu Kα radiation) substantially similar to FIG. 7.28. The pharmaceutical composition of claim 25, wherein the crystallineansolvate of Compound 1 is characterized by an endothermic peak at 95°C.±2° C. as measured by differential scanning calorimetry.